The rational design of ARUK2007145, a dual inhibitor of the α and γ isoforms of the lipid kinase phosphatidylinositol 5-phosphate 4-kinase (PI5P4K).

The phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are therapeutic targets for diseases such as cancer, neurodegeneration and immunological disorders as they are key components in regulating cell signalling pathways. In an effort to make probe molecules available for further exploring these targets, we have previously reported PI5P4Kα-selective and PI5P4Kγ-selective ligands. Herein we report the rational design of PI5P4Kα/γ dual inhibitors, using knowledge gained during the development of selective inhibitors for these proteins. ARUK2007145 (39) is disclosed as a potent, cell-active probe molecule with ADMET properties amenable to conducting experiments in cells.

The Identification of Potent, Selective, and Brain Penetrant PI5P4Kγ Inhibitors as In Vivo-Ready Tool Molecules.

Owing to their central role in regulating cell signaling pathways, the phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are attractive therapeutic targets in diseases such as cancer, neurodegeneration, and immunological disorders. Until now, tool molecules for these kinases have been either limited in potency or isoform selectivity, which has hampered further investigation of biology and drug development. Herein we describe the virtual screening workflow which identified a series of thienylpyrimidines as PI5P4Kγ-selective inhibitors, as well as the medicinal chemistry optimization of this chemotype, to provide potent and selective tool molecules for further use. In vivo pharmacokinetics data are presented for exemplar tool molecules, along with an X-ray structure for ARUK2001607 (15) in complex with PI5P4Kγ, along with its selectivity data against >150 kinases and a Cerep safety panel.

Development of Selective Phosphatidylinositol 5-Phosphate 4-Kinase γ Inhibitors with a Non-ATP-competitive, Allosteric Binding Mode.

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are emerging as attractive therapeutic targets in diseases, such as cancer, immunological disorders, and neurodegeneration, owing to their central role in regulating cell signaling pathways that are either dysfunctional or can be modulated to promote cell survival. Different modes of binding may enhance inhibitor selectivity and reduce off-target effects in cells. Here, we describe efforts to improve the physicochemical properties of the selective PI5P4Kγ inhibitor, NIH-12848 (1). These improvements enabled the demonstration that this chemotype engages PI5P4Kγ in intact cells and that compounds from this series do not inhibit PI5P4Kα or PI5P4Kß. Furthermore, the first X-ray structure of PI5P4Kγ bound to an inhibitor has been determined with this chemotype, confirming an allosteric binding mode. An exemplar from this chemical series adopted two distinct modes of inhibition, including through binding to a putative lipid interaction site which is 18 Å from the ATP pocket.

Controlling Intramolecular Interactions in the Design of Selective, High-Affinity Ligands for the CREBBP Bromodomain.

CREBBP (CBP/KAT3A) and its paralogue EP300 (KAT3B) are lysine acetyltransferases (KATs) that are essential for human development. They each comprise 10 domains through which they interact with >400 proteins, making them important transcriptional co-activators and key nodes in the human protein-protein interactome. The bromodomains of CREBBP and EP300 enable the binding of acetylated lysine residues from histones and a number of other important proteins, including p53, p73, E2F, and GATA1. Here, we report a work to develop a high-affinity, small-molecule ligand for the CREBBP and EP300 bromodomains [(-)-OXFBD05] that shows >100-fold selectivity over a representative member of the BET bromodomains, BRD4(1). Cellular studies using this ligand demonstrate that the inhibition of the CREBBP/EP300 bromodomain in HCT116 colon cancer cells results in lowered levels of c-Myc and a reduction in H3K18 and H3K27 acetylation. In hypoxia (<0.1% O2), the inhibition of the CREBBP/EP300 bromodomain results in the enhanced stabilization of HIF-1α.

Systematic Investigation of the Permeability of Androgen Receptor PROTACs.

Bifunctional molecules known as PROTACs simultaneously bind an E3 ligase and a protein of interest to direct ubiquitination and clearance of that protein, and they have emerged in the past decade as an exciting new paradigm in drug discovery. In order to investigate the permeability and properties of these large molecules, we synthesized two panels of PROTAC molecules, constructed from a range of protein-target ligands, linkers, and E3 ligase ligands. The androgen receptor, which is a well-studied protein in the PROTAC field was used as a model system. The physicochemical properties and permeability of PROTACs are discussed.

Isoxazole-Derived Amino Acids are Bromodomain-Binding Acetyl-Lysine Mimics: Incorporation into Histone H4 Peptides and Histone H3.

A range of isoxazole-containing amino acids was synthesized that displaced acetyl-lysine-containing peptides from the BAZ2A, BRD4(1), and BRD9 bromodomains. Three of these amino acids were incorporated into a histone H4-mimicking peptide and their affinity for BRD4(1) was assessed. Affinities of the isoxazole-containing peptides are comparable to those of a hyperacetylated histone H4-mimicking cognate peptide, and demonstrated a dependence on the position at which the unnatural residue was incorporated. An isoxazole-based alkylating agent was developed to selectively alkylate cysteine residues in situ. Selective monoalkylation of a histone H4-mimicking peptide, containing a lysine to cysteine residue substitution (K12C), resulted in acetyl-lysine mimic incorporation, with high affinity for the BRD4 bromodomain. The same technology was used to alkylate a K18C mutant of histone H3.

Small molecule inhibitors of bromodomain-acetyl-lysine interactions.

Bromodomains are protein modules that bind to acetylated lysine residues. Their interaction with histone proteins suggests that they function as "readers" of histone lysine acetylation, a component of the proposed "histone code". Bromodomain-containing proteins are often found as components of larger protein complexes with roles in fundamental cellular process including transcription. The publication of two potent ligands for the BET bromodomains in 2010 demonstrated that small molecules can inhibit the bromodomain-acetyl-lysine protein-protein interaction. These molecules display strong phenotypic effects in a number of cell lines and affect a range of cancers in vivo. This work stimulated intense interest in developing further ligands for the BET bromodomains and the design of ligands for non-BET bromodomains. Here we review the recent progress in the field with particular attention paid to ligand design, the assays employed in early ligand discovery, and the use of computational approaches to inform ligand design.

Discovery and optimization of small-molecule ligands for the CBP/p300 bromodomains.

Small-molecule inhibitors that target bromodomains outside of the bromodomain and extra-terminal (BET) sub-family are lacking. Here, we describe highly potent and selective ligands for the bromodomain module of the human lysine acetyl transferase CBP/p300, developed from a series of 5-isoxazolyl-benzimidazoles. Our starting point was a fragment hit, which was optimized into a more potent and selective lead using parallel synthesis employing Suzuki couplings, benzimidazole-forming reactions, and reductive aminations. The selectivity of the lead compound against other bromodomain family members was investigated using a thermal stability assay, which revealed some inhibition of the structurally related BET family members. To address the BET selectivity issue, X-ray crystal structures of the lead compound bound to the CREB binding protein (CBP) and the first bromodomain of BRD4 (BRD4(1)) were used to guide the design of more selective compounds. The crystal structures obtained revealed two distinct binding modes. By varying the aryl substitution pattern and developing conformationally constrained analogues, selectivity for CBP over BRD4(1) was increased. The optimized compound is highly potent (Kd = 21 nM) and selective, displaying 40-fold selectivity over BRD4(1). Cellular activity was demonstrated using fluorescence recovery after photo-bleaching (FRAP) and a p53 reporter assay. The optimized compounds are cell-active and have nanomolar affinity for CBP/p300; therefore, they should be useful in studies investigating the biological roles of CBP and p300 and to validate the CBP and p300 bromodomains as therapeutic targets.

A series of potent CREBBP bromodomain ligands reveals an induced-fit pocket stabilized by a cation-π interaction.

The benzoxazinone and dihydroquinoxalinone fragments were employed as novel acetyl lysine mimics in the development of CREBBP bromodomain ligands. While the benzoxazinone series showed low affinity for the CREBBP bromodomain, expansion of the dihydroquinoxalinone series resulted in the first potent inhibitors of a bromodomain outside the BET family. Structural and computational studies reveal that an internal hydrogen bond stabilizes the protein-bound conformation of the dihydroquinoxalinone series. The side chain of this series binds in an induced-fit pocket forming a cation-π interaction with R1173 of CREBBP. The most potent compound inhibits binding of CREBBP to chromatin in U2OS cells.

Progress in the development and application of small molecule inhibitors of bromodomain-acetyl-lysine interactions.

Bromodomains, protein modules that recognize and bind to acetylated lysine, are emerging as important components of cellular machinery. These acetyl-lysine (KAc) "reader" domains are part of the write-read-erase concept that has been linked with the transfer of epigenetic information. By reading KAc marks on histones, bromodomains mediate protein-protein interactions between a diverse array of partners. There has been intense activity in developing potent and selective small molecule probes that disrupt the interaction between a given bromodomain and KAc. Rapid success has been achieved with the BET family of bromodomains, and a number of potent and selective probes have been reported. These compounds have enabled linking of the BET bromodomains with diseases, including cancer and inflammation, suggesting that bromodomains are druggable targets. Herein, we review the biology of the bromodomains and discuss the SAR for the existing small molecule probes. The biology that has been enabled by these compounds is summarized.