Design and synthesis of aminopyridine containing biaryls reducing c-MYC protein levels in cells.

The c-MYC oncogene transcription factor has been implicated in cell cycle regulation controlling cell growth and proliferation. It is tightly regulated in normal cells, but has been shown to be deregulated in cancer cells, and is thus an attractive target for oncogenic therapies. Building upon previous SAR, a series of analogues containing benzimidazole core replacements were prepared and evaluated, leading to the identification of imidazopyridazine compounds that were shown to possess equivalent or improved c-MYC HTRF pEC50 values, lipophilicity, solubility, and rat pharmacokinetics. The imidazopyridazine core was therefore determined to be superior to the original benzimidazole core and a viable alternate for continued lead optimization and medicinal chemistry campaigns.

Allosteric Wip1 phosphatase inhibition through flap-subdomain interaction.

Although therapeutic interventions of signal-transduction cascades with targeted kinase inhibitors are a well-established strategy, drug-discovery efforts to identify targeted phosphatase inhibitors have proven challenging. Herein we report a series of allosteric, small-molecule inhibitors of wild-type p53-induced phosphatase (Wip1), an oncogenic phosphatase common to multiple cancers. Compound binding to Wip1 is dependent on a 'flap' subdomain located near the Wip1 catalytic site that renders Wip1 structurally divergent from other members of the protein phosphatase 2C (PP2C) family and that thereby confers selectivity for Wip1 over other phosphatases. Treatment of tumor cells with the inhibitor GSK2830371 increases phosphorylation of Wip1 substrates and causes growth inhibition in both hematopoietic tumor cell lines and Wip1-amplified breast tumor cells harboring wild-type TP53. Oral administration of Wip1 inhibitors in mice results in expected pharmacodynamic effects and causes inhibition of lymphoma xenograft growth. To our knowledge, GSK2830371 is the first orally active, allosteric inhibitor of Wip1 phosphatase.

Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1α Endoribonuclease Activity.

Activation of the inositol-requiring enzyme-1 alpha (IRE1α) protein caused by endoplasmic reticulum stress results in the homodimerization of the N-terminal endoplasmic reticulum luminal domains, autophosphorylation of the cytoplasmic kinase domains, and conformational changes to the cytoplasmic endoribonuclease (RNase) domains, which render them functional and can lead to the splicing of X-box binding protein 1 (XBP 1) mRNA. Herein, we report the first crystal structures of the cytoplasmic portion of a human phosphorylated IRE1α dimer in complex with (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide, a novel, IRE1α-selective kinase inhibitor, and staurosporine, a broad spectrum kinase inhibitor. (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide inhibits both the kinase and RNase activities of IRE1α. The inhibitor interacts with the catalytic residues Lys599 and Glu612 and displaces the kinase activation loop to the DFG-out conformation. Inactivation of IRE1α RNase activity appears to be caused by a conformational change, whereby the αC helix is displaced, resulting in the rearrangement of the kinase domain-dimer interface and a rotation of the RNase domains away from each other. In contrast, staurosporine binds at the ATP-binding site of IRE1α, resulting in a dimer consistent with RNase active yeast Ire1 dimers. Activation of IRE1α RNase activity appears to be promoted by a network of hydrogen bond interactions between highly conserved residues across the RNase dimer interface that place key catalytic residues poised for reaction. These data implicate that the intermolecular interactions between conserved residues in the RNase domain are required for activity, and that the disruption of these interactions can be achieved pharmacologically by small molecule kinase domain inhibitors.

Structural characterization of dicyanopyridine containing DNMT1-selective, non-nucleoside inhibitors.

DNMT1 maintains the parental DNA methylation pattern on newly replicated hemimethylated DNA. The failure of this maintenance process causes aberrant DNA methylation that affects transcription and contributes to the development and progression of cancers such as acute myeloid leukemia. Here, we structurally characterized a set of newly discovered DNMT1-selective, reversible, non-nucleoside inhibitors that bear a core 3,5-dicyanopyridine moiety, as exemplified by GSK3735967, to better understand their mechanism of inhibition. All of the dicyanopydridine-containing inhibitors examined intercalate into the hemimethylated DNA between two CpG base pairs through the DNA minor groove, resulting in conformational movement of the DNMT1 active-site loop. In addition, GSK3735967 introduces two new binding sites, where it interacts with and stabilizes the displaced DNMT1 active-site loop and it occupies an open aromatic cage in which trimethylated histone H4 lysine 20 is expected to bind. Our work represents a substantial step in generating potent, selective, and non-nucleoside inhibitors of DNMT1.

Discovery of Proline-Based p300/CBP Inhibitors Using DNA-Encoded Library Technology in Combination with High-Throughput Screening.

E1A binding protein (p300) and CREB binding protein (CBP) are two highly homologous and multidomain histone acetyltransferases. These two proteins are involved in many cellular processes by acting as coactivators of a large number of transcription factors. Dysregulation of p300/CBP has been found in a variety of cancers and other diseases, and inhibition has been shown to decrease Myc expression. Herein, we report the identification of a series of highly potent, proline-based small-molecule p300/CBP histone acetyltransferase (HAT) inhibitors using DNA-encoded library technology in combination with high-throughput screening. The strategy of reducing ChromlogD and fluorination of metabolic soft spots was explored to improve the pharmacokinetic properties of potent p300 inhibitors. Fluorination of both cyclobutyl and proline rings of 22 led to not only reduced clearance but also improved cMyc cellular potency.

Cell-Based Drug Discovery: Identification and Optimization of Small Molecules that Reduce c-MYC Protein Levels in Cells.

Elevated expression of the c-MYC oncogene is one of the most common abnormalities in human cancers. Unfortunately, efforts to identify pharmacological inhibitors that directly target MYC have not yet yielded a drug-like molecule due to the lack of any known small molecule binding pocket in the protein, which could be exploited to disrupt MYC function. We have recently described a strategy to target MYC indirectly, where a screening effort designed to identify compounds that can rapidly decrease endogenous c-MYC protein levels in a MYC-amplified cell line led to the discovery of a compound series that phenocopies c-MYC knockdown by siRNA. Herein, we describe our medicinal chemistry program that led to the discovery of potent, orally bioavailable c-MYC-reducing compounds. The development of a minimum pharmacophore model based on empirical structure activity relationship as well as the property-based approach used to modulate pharmacokinetics properties will be highlighted.

2,3,5-Trisubstituted pyridines as selective AKT inhibitors. Part II: Improved drug-like properties and kinase selectivity from azaindazoles.

A novel series of AKT inhibitors containing 2,3,5-trisubstituted pyridines with novel azaindazoles as hinge binding elements are described. Among these, the 4,7-diazaindazole compound 2c has improved drug-like properties and kinase selectivity than those of indazole 1, and displays greater than 80% inhibition of GSK3beta phosphorylation in a BT474 tumor xenograft model in mice.

2,3,5-Trisubstituted pyridines as selective AKT inhibitors-Part I: Substitution at 2-position of the core pyridine for ROCK1 selectivity.

2,3,5-Trisubstituted pyridines have been designed as potent AKT inhibitors that are selective against ROCK1 based on the comparison between AKT and ROCK1 structures. Substitution at the 2-position of the core pyridine is the key element to provide selectivity against ROCK1. An X-ray co-crystal structure of 9p in PKA supports the proposed rationale of ROCK1 selectivity.

Tetrasubstituted pyridines as potent and selective AKT inhibitors: Reduced CYP450 and hERG inhibition of aminopyridines.

The synthesis and evaluation of tetrasubstituted aminopyridines, bearing novel azaindazole hinge binders, as potent AKT inhibitors are described. Compound 14c was identified as a potent AKT inhibitor that demonstrated reduced CYP450 inhibition and an improved developability profile compared to those of previously described trisubstituted pyridines. It also displayed dose-dependent inhibition of both phosphorylation of GSK3beta and tumor growth in a BT474 tumor xenograft model in mice.

Discovery of Isoxazole Amides as Potent and Selective SMYD3 Inhibitors.

We report herein the discovery of isoxazole amides as potent and selective SET and MYND Domain-Containing Protein 3 (SMYD3) inhibitors. Elucidation of the structure-activity relationship of the high-throughput screening (HTS) lead compound 1 provided potent and selective SMYD3 inhibitors. The SAR optimization, cocrystal structures of small molecules with SMYD3, and mode of inhibition (MOI) characterization of compounds are described. The synthesis and biological and pharmacokinetic profiles of compounds are also presented.

Discovery of 5-pyrrolopyridinyl-2-thiophenecarboxamides as potent AKT kinase inhibitors.

A pyrrolopyridinyl thiophene carboxamide 7 was discovered as a tractable starting point for a lead optimization effort in an AKT kinase inhibition program. SAR studies aided by a co-crystal structure of 7 in AKT2 led to the identification of AKT inhibitors with subnanomolar potency. Representative compounds showed antiproliferative activity as well as inhibition of phosphorylation of the downstream target GSK3beta.

Aminofurazans as potent inhibitors of AKT kinase.

AKT inhibitors containing an imidazopyridine aminofurazan scaffold have been optimized. We have previously disclosed identification of the AKT inhibitor GSK690693, which has been evaluated in clinical trials in cancer patients. Herein we describe recent efforts focusing on investigating a distinct region of this scaffold that have afforded compounds (30 and 32) with comparable activity profiles to that of GSK690693.

Generation and Validation of Intracellular Ubiquitin Variant Inhibitors for USP7 and USP10.

Post-translational modification of the p53 signaling pathway plays an important role in cell cycle progression and stress-induced apoptosis. Indeed, a large body of work has shown that dysregulation of p53 and its E3 ligase MDM2 by the ubiquitin-proteasome system (UPS) promotes carcinogenesis and malignant transformation. Thus, drug discovery efforts have focused on the restoration of wild-type p53 activity or inactivation of oncogenic mutant p53 by targeted inhibition of UPS components, particularly key deubiquitinases (DUBs) of the ubiquitin-specific protease (USP) class. However, development of selective small-molecule USP inhibitors has been challenging, partly due to the highly conserved structural features of the catalytic sites across the class. To tackle this problem, we devised a protein engineering strategy for rational design of inhibitors for DUBs and other UPS proteins. We employed a phage-displayed ubiquitin variant (UbV) library to develop inhibitors targeting the DUBs USP7 and USP10, which are involved in regulating levels of p53 and MDM2. We were able to identify UbVs that bound USP7 or USP10 with high affinity and inhibited deubiquitination activity. We solved the crystal structure of UbV.7.2 and rationalized the molecular basis for enhanced affinity and specificity for USP7. Finally, cell death was increased significantly by UbV.7.2 expression in a colon cancer cell line that was treated with the chemotherapy drug cisplatin, demonstrating the therapeutic potential of inhibiting USP7 by this approach.

Discovery of aminopyridine-based inhibitors of bacterial enoyl-ACP reductase (FabI).

Bacterial enoyl-ACP reductase (FabI) catalyzes the final step in each cycle of bacterial fatty acid biosynthesis and is an attractive target for the development of new antibacterial agents. Our efforts to identify potent, selective FabI inhibitors began with screening of the GlaxoSmithKline proprietary compound collection, which identified several small-molecule inhibitors of Staphylococcus aureus FabI. Through a combination of iterative medicinal chemistry and X-ray crystal structure based design, one of these leads was developed into the novel aminopyridine derivative 9, a low micromolar inhibitor of FabI from S. aureus (IC(50) = 2.4 microM) and Haemophilus influenzae (IC(50) = 4.2 microM). Compound 9 has good in vitro antibacterial activity against several organisms, including S. aureus (MIC = 0.5 microg/mL), and is effective in vivo in a S. aureus groin abscess infection model in rats. Through FabI overexpressor and macromolecular synthesis studies, the mode of action of 9 has been confirmed to be inhibition of fatty acid biosynthesis via inhibition of FabI. Taken together, these results support FabI as a valid antibacterial target and demonstrate the potential of small-molecule FabI inhibitors for the treatment of bacterial infections.

Discovery of novel AKT inhibitors with enhanced anti-tumor effects in combination with the MEK inhibitor.

Tumor cells upregulate many cell signaling pathways, with AKT being one of the key kinases to be activated in a variety of malignancies. GSK2110183 and GSK2141795 are orally bioavailable, potent inhibitors of the AKT kinases that have progressed to human clinical studies. Both compounds are selective, ATP-competitive inhibitors of AKT 1, 2 and 3. Cells treated with either compound show decreased phosphorylation of several substrates downstream of AKT. Both compounds have desirable pharmaceutical properties and daily oral dosing results in a sustained inhibition of AKT activity as well as inhibition of tumor growth in several mouse tumor models of various histologic origins. Improved kinase selectivity was associated with reduced effects on glucose homeostasis as compared to previously reported ATP-competitive AKT kinase inhibitors. In a diverse cell line proliferation screen, AKT inhibitors showed increased potency in cell lines with an activated AKT pathway (via PI3K/PTEN mutation or loss) while cell lines with activating mutations in the MAPK pathway (KRAS/BRAF) were less sensitive to AKT inhibition. Further investigation in mouse models of KRAS driven pancreatic cancer confirmed that combining the AKT inhibitor, GSK2141795 with a MEK inhibitor (GSK2110212; trametinib) resulted in an enhanced anti-tumor effect accompanied with greater reduction in phospho-S6 levels. Taken together these results support clinical evaluation of the AKT inhibitors in cancer, especially in combination with MEK inhibitor.

Discovery of GSK2656157: An Optimized PERK Inhibitor Selected for Preclinical Development.

We recently reported the discovery of GSK2606414 (1), a selective first in class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), which inhibited PERK activation in cells and demonstrated tumor growth inhibition in a human tumor xenograft in mice. In continuation of our drug discovery program, we applied a strategy to decrease inhibitor lipophilicity as a means to improve physical properties and pharmacokinetics. This report describes our medicinal chemistry optimization culminating in the discovery of the PERK inhibitor GSK2656157 (6), which was selected for advancement to preclinical development.

Discovery of 7-methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), a potent and selective first-in-class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK).

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is activated in response to a variety of endoplasmic reticulum stresses implicated in numerous disease states. Evidence that PERK is implicated in tumorigenesis and cancer cell survival stimulated our search for small molecule inhibitors. Through screening and lead optimization using the human PERK crystal structure, we discovered compound 38 (GSK2606414), an orally available, potent, and selective PERK inhibitor. Compound 38 inhibits PERK activation in cells and inhibits the growth of a human tumor xenograft in mice.

Structure-based design of potent and selective 3-phosphoinositide-dependent kinase-1 (PDK1) inhibitors.

Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.

Aminoindazole PDK1 Inhibitors: A Case Study in Fragment-Based Drug Discovery.

Fragment screening of phosphoinositide-dependent kinase-1 (PDK1) in a biochemical kinase assay afforded hits that were characterized and prioritized based on ligand efficiency and binding interactions with PDK1 as determined by NMR. Subsequent crystallography and follow-up screening led to the discovery of aminoindazole 19, a potent leadlike PDK1 inhibitor with high ligand efficiency. Well-defined structure-activity relationships and protein crystallography provide a basis for further elaboration and optimization of 19 as a PDK1 inhibitor.

Characterization of an Akt kinase inhibitor with potent pharmacodynamic and antitumor activity.

Akt kinases 1, 2, and 3 are important regulators of cell survival and have been shown to be constitutively active in a variety of human tumors. GSK690693 is a novel ATP-competitive, low-nanomolar pan-Akt kinase inhibitor. It is selective for the Akt isoforms versus the majority of kinases in other families; however, it does inhibit additional members of the AGC kinase family. It causes dose-dependent reductions in the phosphorylation state of multiple proteins downstream of Akt, including GSK3 beta, PRAS40, and Forkhead. GSK690693 inhibited proliferation and induced apoptosis in a subset of tumor cells with potency consistent with intracellular inhibition of Akt kinase activity. In immune-compromised mice implanted with human BT474 breast carcinoma xenografts, a single i.p. administration of GSK690693 inhibited GSK3 beta phosphorylation in a dose- and time-dependent manner. After a single dose of GSK690693, >3 micromol/L drug concentration in BT474 tumor xenografts correlated with a sustained decrease in GSK3 beta phosphorylation. Consistent with the role of Akt in insulin signaling, treatment with GSK690693 resulted in acute and transient increases in blood glucose level. Daily administration of GSK690693 produced significant antitumor activity in mice bearing established human SKOV-3 ovarian, LNCaP prostate, and BT474 and HCC-1954 breast carcinoma xenografts. Immunohistochemical analysis of tumor xenografts after repeat dosing with GSK690693 showed reductions in phosphorylated Akt substrates in vivo. These results support further evaluation of GSK690693 as an anticancer agent.