Discovery of IPN60090, a Clinical Stage Selective Glutaminase-1 (GLS-1) Inhibitor with Excellent Pharmacokinetic and Physicochemical Properties.

Inhibition of glutaminase-1 (GLS-1) hampers the proliferation of tumor cells reliant on glutamine. Known glutaminase inhibitors have potential limitations, and in vivo exposures are potentially limited due to poor physicochemical properties. We initiated a GLS-1 inhibitor discovery program focused on optimizing physicochemical and pharmacokinetic properties, and have developed a new selective inhibitor, compound 27 (IPN60090), which is currently in phase 1 clinical trials. Compound 27 attains high oral exposures in preclinical species, with strong in vivo target engagement, and should robustly inhibit glutaminase in humans.

Discovery and in Vivo Evaluation of Macrocyclic Mcl-1 Inhibitors Featuring an α-Hydroxy Phenylacetic Acid Pharmacophore or Bioisostere.

Overexpression of the antiapoptotic protein Mcl-1 provides a survival advantage to some cancer cells, making inhibition of this protein an attractive therapeutic target for the treatment of certain types of tumors. Herein, we report our efforts toward the identification of a novel series of macrocyclic Mcl-1 inhibitors featuring an α-hydroxy phenylacetic acid pharmacophore or bioisostere. This work led to the discovery of 1, a potent Mcl-1 inhibitor (IC50 = 19 nM in an OPM-2 cell viability assay) with good pharmacokinetic properties and excellent in vivo efficacy in an OPM-2 multiple myeloma xenograft model.

Prospective discovery of small molecule enhancers of an E3 ligase-substrate interaction.

Protein-protein interactions (PPIs) governing the recognition of substrates by E3 ubiquitin ligases are critical to cellular function. There is significant therapeutic potential in the development of small molecules that modulate these interactions; however, rational design of small molecule enhancers of PPIs remains elusive. Herein, we report the prospective identification and rational design of potent small molecules that enhance the interaction between an oncogenic transcription factor, ß-Catenin, and its cognate E3 ligase, SCFß-TrCP. These enhancers potentiate the ubiquitylation of mutant ß-Catenin by ß-TrCP in vitro and induce the degradation of an engineered mutant ß-Catenin in a cellular system. Distinct from PROTACs, these drug-like small molecules insert into a naturally occurring PPI interface, with contacts optimized for both the substrate and ligase within the same small molecule entity. The prospective discovery of 'molecular glue' presented here provides a paradigm for the development of small molecule degraders targeting hard-to-drug proteins.

AMG 176, a Selective MCL1 Inhibitor, Is Effective in Hematologic Cancer Models Alone and in Combination with Established Therapies.

The prosurvival BCL2 family member MCL1 is frequently dysregulated in cancer. To overcome the significant challenges associated with inhibition of MCL1 protein-protein interactions, we rigorously applied small-molecule conformational restriction, which culminated in the discovery of AMG 176, the first selective MCL1 inhibitor to be studied in humans. We demonstrate that MCL1 inhibition induces a rapid and committed step toward apoptosis in subsets of hematologic cancer cell lines, tumor xenograft models, and primary patient samples. With the use of a human MCL1 knock-in mouse, we demonstrate that MCL1 inhibition at active doses of AMG 176 is tolerated and correlates with clear pharmacodynamic effects, demonstrated by reductions in B cells, monocytes, and neutrophils. Furthermore, the combination of AMG 176 and venetoclax is synergistic in acute myeloid leukemia (AML) tumor models and in primary patient samples at tolerated doses. These results highlight the therapeutic promise of AMG 176 and the potential for combinations with other BH3 mimetics. SIGNIFICANCE: AMG 176 is a potent, selective, and orally bioavailable MCL1 inhibitor that induces a rapid commitment to apoptosis in models of hematologic malignancies. The synergistic combination of AMG 176 and venetoclax demonstrates robust activity in models of AML at tolerated doses, highlighting the promise of BH3-mimetic combinations in hematologic cancers.See related commentary by Leber et al., p. 1511.This article is highlighted in the In This Issue feature, p. 1494.

Discovery and in Vivo Evaluation of the Potent and Selective PI3Kδ Inhibitors 2-((1S)-1-((6-Amino-5-cyano-4-pyrimidinyl)amino)ethyl)-6-fluoro-N-methyl-3-(2-pyridinyl)-4-quinolinecarboxamide (AM-0687) and 2-((1S)-1-((6-Amino-5-cyano-4-pyrimidinyl)amino)ethyl)-5-fluoro-N-methyl-3-(2-pyridinyl)-4-quinolinecarboxamide (AM-1430).

Optimization of the potency and pharmacokinetic profile of 2,3,4-trisubstituted quinoline, 4, led to the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 6a (AM-0687) and 7 (AM-1430). On the basis of their improved profile, these analogs were selected for in vivo pharmacodynamic (PD) and efficacy experiments in animal models of inflammation. The in vivo PD studies, which were carried out in a mouse pAKT inhibition animal model, confirmed the observed potency of 6a and 7 in biochemical and cellular assays. Efficacy experiments in a keyhole limpet hemocyanin model in rats demonstrated that administration of either 6a or 7 resulted in a strong dose-dependent reduction of IgG and IgM specific antibodies. The excellent in vitro and in vivo profiles of these analogs make them suitable for further development.

Identification of potent and selective MTH1 inhibitors.

Structure based design of a novel class of aminopyrimidine MTH1 (MutT homolog 1) inhibitors is described. Optimization led to identification of IACS-4759 (compound 5), a sub-nanomolar inhibitor of MTH1 with excellent cell permeability and good metabolic stability in microsomes. This compound robustly inhibited MTH1 activity in cells and proved to be an excellent tool for interrogation of the utility of MTH1 inhibition in the context of oncology.

Observed bromodomain flexibility reveals histone peptide- and small molecule ligand-compatible forms of ATAD2.

Preventing histone recognition by bromodomains emerges as an attractive therapeutic approach in cancer. Overexpression of ATAD2 (ATPase family AAA domain-containing 2 isoform A) in cancer cells is associated with poor prognosis making the bromodomain of ATAD2 a promising epigenetic therapeutic target. In the development of an in vitro assay and identification of small molecule ligands, we conducted structure-guided studies which revealed a conformationally flexible ATAD2 bromodomain. Structural studies on apo-, peptide-and small molecule-ATAD2 complexes (by co-crystallization) revealed that the bromodomain adopts a 'closed', histone-compatible conformation and a more 'open' ligand-compatible conformation of the binding site respectively. An unexpected conformational change of the conserved asparagine residue plays an important role in driving the peptide-binding conformation remodelling. We also identified dimethylisoxazole-containing ligands as ATAD2 binders which aided in the validation of the in vitro screen and in the analysis of these conformational studies.

Discovery and in vivo evaluation of (S)-N-(1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine (AMG319) and related PI3Kδ inhibitors for inflammation and autoimmune disease.

The development and optimization of a series of quinolinylpurines as potent and selective PI3Kδ kinase inhibitors with excellent physicochemical properties are described. This medicinal chemistry effort led to the identification of 1 (AMG319), a compound with an IC50 of 16 nM in a human whole blood assay (HWB), excellent selectivity over a large panel of protein kinases, and a high level of in vivo efficacy as measured by two rodent disease models of inflammation.

Trifluoromethyl group as a pharmacophore: effect of replacing a CF3 group on binding and agonist activity of a glucocorticoid receptor ligand.

Compound 1, a potent glucocorticoid receptor ligand, contains a quaternary carbon bearing trifluoromethyl and hydroxyl groups. This paper describes the effect of replacing the trifluoromethyl group on binding and agonist activity of the GR ligand 1. The results illustrate that replacing the CF3 group with a cyclohexylmethyl or benzyl group maintains the GR binding potency. These substitutions alter the functional behavior of the GR ligands from agonists to antagonists. Docking studies suggest that the benzyl analog 19 binds in a similar fashion as the GR antagonist, RU486. The central benzyl group of 19 and the C-11 dimethylaniline moiety of RU486 overlay. Binding of compound 19 is believed to force helix 12 to adopt an open conformation and this leads to the antagonist properties of the non-CF3 ligands carrying a large group at the center of the molecule.

Optimization of 2-phenylaminoimidazo[4,5-h]isoquinolin-9-ones: orally active inhibitors of lck kinase.

The tyrosine kinase p56lck (lck) is essential for T cell activation; thus, inhibitors of lck have potential utility as autoimmune agents. Our initial disclosure of a new class of lck inhibitors based on the phenylaminoimidazoisoquinolin-9-one showed reasonable cellular activity but did not work in vivo upon oral administration. Our current work highlights the further use of rational drug design and molecular modeling to produce a series of lck inhibitors that demonstrate cellular activity below 100 nM and are as efficacious as cyclosporin A in an in vivo mouse model of anti-CD3-induced IL-2 production.

Discovery of 2-phenylamino-imidazo[4,5-h]isoquinolin-9-ones: a new class of inhibitors of lck kinase.

An imidazo[4,5-h]isoquinolin-7,9-dione (1) was identified as an adenosine 5'-triphosphate competitive inhibitor of lck by high throughput screening. Initial structure-activity relationship studies identified the dichlorophenyl ring and the imide NH as important pharmacophores. A binding model was constructed to understand how 1 binds to a related kinase, hck. These results suggested that removing the gem-dimethyl group and flattening the ring would enhance activity. This was realized by converting 1 to the imidazo[4,5-h]isoquinolin-9-one (20), resulting in an 18-fold improvement in potency against lck and a 50-fold increase in potency in a cellular assay.