The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P) is emerging as a key regulator of lipid transfer in ER-membrane contact sites. Four different phosphatidylinositol 4-kinase (PI4K) enzymes generate PI4P in different membrane compartments supporting distinct cellular processes, many of which are crucial for the maintenance of cellular integrity but also hijacked by intracellular pathogens. While type III PI4Ks have been targeted by small molecular inhibitors, thus helping decipher their importance in cellular physiology, no inhibitors are available for the type II PI4Ks, which hinders investigations into their cellular functions. Here we describe the identification of small molecular inhibitors of PI4K2A by implementing a large scale small molecule high throughput screening. A novel assay was developed that allows testing of selected inhibitors against PI4K2A in intact cells using a BRET approach adapted to plate readers. The compounds disclosed here will pave the way to the optimization of PI4K2A inhibitors that can be used in cellular and animal studies to better understand the role of this enzyme in both normal and pathological states.

Resolution of acute inflammation is governed, in part, by specialized pro-resolving mediators, including lipoxins, resolvins, protectins, and maresins. Among them, resolvin D1 (RvD1) exhibits potent pro-resolving functions via activating human resolvin D1 receptor (DRV1/GPR32). RvD1 is a complex molecule that requires challenging organic synthesis, diminishing its potential as a therapeutic. Therefore, we implemented a high-throughput screening of small-molecule libraries and identified several chemotypes that activated recombinant DRV1, represented by NCGC00120943 (C1A), NCGC00135472 (C2A), pMPPF, and pMPPI. These chemotypes also elicited rapid impedance changes in cells overexpressing recombinant DRV1. With human macrophages, they each stimulated phagocytosis of serum-treated zymosan at concentrations comparable with that of RvD1, the endogenous DRV1 ligand. In addition, macrophage phagocytosis of live E. coli was significantly increased by these chemotypes in DRV1-transfected macrophages, compared with mock-transfected cells. Taken together, these chemotypes identified by unbiased screens act as RvD1 mimetics, exhibiting pro-resolving functions via interacting with human DRV1.

Brain injury and inflammation induces a local release of extracellular vesicles (EVs) from astrocytes carrying proteins, RNAs, and microRNAs into the circulation. When these vesicles reach the liver, they stimulate the secretion of cytokines that mobilize peripheral immune cell infiltration into the brain, which can cause secondary tissue damage and impair recovery. Recent studies suggest that suppression of EV biosynthesis through neutral sphingomyelinase 2 (nSMase2) inhibition may represent a new therapeutic strategy. Unfortunately, currently available nSMase2 inhibitors exhibit low potency (IC50 ≥ 1 μM), poor solubility and/or limited brain penetration. Through a high throughput screening campaign of >365,000 compounds against human nSMase2 we identified 2,6-Dimethoxy-4-(5-Phenyl-4-Thiophen-2-yl-1H-Imidazol-2-yl)-Phenol (DPTIP), a potent (IC50 30 nM), selective, metabolically stable, and brain penetrable (AUCbrain/AUCplasma = 0.26) nSMase2 inhibitor. DPTIP dose-dependently inhibited EV release in primary astrocyte cultures. In a mouse model of brain injury conducted in GFAP-GFP mice, DPTIP potently (10 mg/kg IP) inhibited IL-1β-induced astrocyte-derived EV release (51 ± 13%; p < 0.001). This inhibition led to a reduction of cytokine upregulation in liver and attenuation of the infiltration of immune cells into the brain (80 ± 23%; p < 0.01). A structurally similar but inactive analog had no effect in vitro or in vivo.

A dose responsive quantitative high throughput screen (qHTS) of >350,000 compounds against a human relaxin/insulin-like family peptide receptor (RXFP1) transfected HEK293 cell line identified 2-acetamido-N-phenylbenzamides 1 and 3 with modest agonist activity. An extensive structure-activity study has been undertaken to optimize the potency, efficacy, and physical properties of the series, resulting in the identification of compound 65 (ML-290), which has excellent in vivo PK properties with high levels of systemic exposure. This series, exemplified by 65, has produced first-in-class small-molecule agonists of RXFP1 and is a potent activator of anti-fibrotic genes.

Recently, we showed that TSH-enhanced differentiation of a human preosteoblast-like cell model involved a β-arrestin 1 (β-Arr 1)-mediated pathway. To study this pathway in more detail, we sought to discover a small molecule ligand that was functionally selective toward human TSH receptor (TSHR) activation of β-Arr 1. High-throughput screening using a cell line stably expressing mutated TSHRs and mutated β-Arr 1 (DiscoverX1 cells) led to the discovery of agonists that stimulated translocation of β-Arr 1 to the TSHR, but did not activate Gs-mediated signaling pathways, i.e., cAMP production. D3-βArr (NCGC00379308) was selected. In DiscoverX1 cells, D3-βArr stimulated β-Arr 1 translocation with a 5.1-fold greater efficacy than TSH and therefore potentiated the effect of TSH in stimulating β-Arr 1 translocation. In human U2OS-TSHR cells expressing wild-type TSHRs, which is a model of human preosteoblast-like cells, TSH upregulated the osteoblast-specific genes osteopontin (OPN) and alkaline phosphatase (ALPL). D3-βArr alone had only a weak effect to upregulate these bone markers, but D3-βArr potentiated TSH-induced upregulation of ALPL and OPN mRNA levels 1.6-fold and 5.5-fold, respectively, at the maximum dose of ligands. Furthermore, the positive allosteric modulator effect of D3-βArr resulted in an increase of TSH-induced secretion of OPN protein. In summary, we have discovered the first small molecule positive allosteric modulator of TSHR. As D3-βArr potentiates the effect of TSH to enhance differentiation of a human preosteoblast in an in vitro model, it will allow a novel experimental approach for probing the role of TSH-induced β-Arr 1 signaling in osteoblast differentiation.

Akt plays a major role in tumorigenesis and the development of specific Akt inhibitors as effective cancer therapeutics has been challenging. Here, we report the identification of a highly specific allosteric inhibitor of Akt through a FRET-based high-throughput screening, and characterization of its inhibitory mechanism. Out of 373,868 compounds screened, 4-phenylquinolin-2(1H)-one specifically decreased Akt phosphorylation at both T308 and S473, and inhibited Akt kinase activity (IC50 = 6 µM) and downstream signaling. 4-Phenylquinolin-2(1H)-one did not alter the activity of upstream kinases including PI3K, PDK1, and mTORC2 as well as closely related kinases that affect cell proliferation and survival such as SGK1, PKA, PKC, or ERK1/2. This compound inhibited the proliferation of cancer cells but displayed less toxicity compared to inhibitors of PI3K or mTOR. Kinase profiling efforts revealed that 4-phenylquinolin-2(1H)-one does not bind to the kinase active site of over 380 human kinases including Akt. However, 4-phenylquinolin-2(1H)-one interacted with the PH domain of Akt, apparently inducing a conformation that hinders S473 and T308 phosphorylation by mTORC2 and PDK1. In conclusion, we demonstrate that 4-phenylquinolin-2(1H)-one is an exquisitely selective Akt inhibitor with a distinctive molecular mechanism, and a promising lead compound for further optimization toward the development of novel cancer therapeutics.

Infection with human cytomegalovirus (HCMV) is a threat for pregnant women and immunocompromised hosts. Although limited drugs are available, development of new agents against HCMV is desired. Through screening of the LOPAC library, we identified emetine as HCMV inhibitor. Additional studies confirmed its anti-HCMV activities in human foreskin fibroblasts: EC50-40±1.72 nM, CC50-8±0.56 μM, and selectivity index of 200. HCMV inhibition occurred after virus entry, but before DNA replication, and resulted in decreased expression of viral proteins. Synergistic virus inhibition was achieved when emetine was combined with ganciclovir. In a mouse CMV (MCMV) model, emetine was well-tolerated, displayed long half-life, preferential distribution to tissues over plasma, and effectively suppressed MCMV. Since the in vitro anti-HCMV activity of emetine decreased significantly in low-density cells, a mechanism involving cell cycle regulation was suspected. HCMV inhibition by emetine depended on ribosomal processing S14 (RPS14) binding to MDM2, leading to disruption of HCMV-induced MDM2-p53 and MDM2-IE2 interactions. Irrespective of cell density, emetine induced RPS14 translocation into the nucleus during infection. In infected high-density cells, MDM2 was available for interaction with RPS14, resulting in disruption of MDM2-p53 interaction. However, in low-density cells the pre-existing interaction of MDM2-p53 could not be disrupted, and RPS14 could not interact with MDM2. In high-density cells the interaction of MDM2-RPS14 resulted in ubiquitination and degradation of RPS14, which was not observed in low-density cells. In infected-only or in non-infected emetine-treated cells, RPS14 failed to translocate into the nucleus, hence could not interact with MDM2, and was not ubiquitinated. HCMV replicated similarly in RPS14 knockdown or control cells, but emetine did not inhibit virus replication in the former cell line. The interaction of MDM2-p53 was maintained in infected RPS14 knockdown cells despite emetine treatment, confirming a unique mechanism by which emetine exploits RPS14 to disrupt MDM2-p53 interaction. Summarized, emetine may represent a promising candidate for HCMV therapy alone or in combination with ganciclovir through a novel host-dependent mechanism.

The GPCR relaxin family peptide receptor 1 (RXFP1) mediates the action of relaxin peptide hormone, including its tissue remodeling and antifibrotic effects. The peptide has a short half-life in plasma, limiting its therapeutic utility. However, small-molecule agonists of human RXFP1 can overcome this limitation and may provide a useful therapeutic approach, especially for chronic diseases such as heart failure and fibrosis. The first small-molecule agonists of RXFP1 were recently identified from a high-throughput screening, using a homogeneous cell-based cAMP assay. Optimization of the hit compounds resulted in a series of highly potent and RXFP1 selective agonists with low cytotoxicity, and excellent in vitro ADME and pharmacokinetic properties. Here, we undertook extensive site-directed mutagenesis studies in combination with computational modeling analysis to probe the molecular basis of the small-molecule binding to RXFP1. The results showed that the agonists bind to an allosteric site of RXFP1 in a manner that closely interacts with the seventh transmembrane domain (TM7) and the third extracellular loop (ECL3). Several residues were determined to play an important role in the agonist binding and receptor activation, including a hydrophobic region at TM7 consisting of W664, F668, and L670. The G659/T660 motif within ECL3 is crucial to the observed species selectivity of the agonists for RXFP1. The receptor binding and activation effects by the small molecule ML290 were compared with the cognate ligand, relaxin, providing valuable insights on the structural basis and molecular mechanism of receptor activation and selectivity for RXFP1.

A high-throughput screening campaign was conducted to interrogate a 380,000+ small-molecule library for novel D2 dopamine receptor modulators using a calcium mobilization assay. Active agonist compounds from the primary screen were examined for orthogonal D2 dopamine receptor signaling activities including cAMP modulation and β-arrestin recruitment. Although the majority of the subsequently confirmed hits activated all signaling pathways tested, several compounds showed a diminished ability to stimulate β-arrestin recruitment. One such compound (MLS1547; 5-chloro-7-[(4-pyridin-2-ylpiperazin-1-yl)methyl]quinolin-8-ol) is a highly efficacious agonist at D2 receptor-mediated G protein-linked signaling, but does not recruit β-arrestin as demonstrated using two different assays. This compound does, however, antagonize dopamine-stimulated β-arrestin recruitment to the D2 receptor. In an effort to investigate the chemical scaffold of MLS1547 further, we characterized a set of 24 analogs of MLS1547 with respect to their ability to inhibit cAMP accumulation or stimulate β-arrestin recruitment. A number of the analogs were similar to MLS1547 in that they displayed agonist activity for inhibiting cAMP accumulation, but did not stimulate β-arrestin recruitment (i.e., they were highly biased). In contrast, other analogs displayed various degrees of G protein signaling bias. These results provided the basis to use pharmacophore modeling and molecular docking analyses to build a preliminary structure-activity relationship of the functionally selective properties of this series of compounds. In summary, we have identified and characterized a novel G protein-biased agonist of the D2 dopamine receptor and identified structural features that may contribute to its biased signaling properties.

Eya proteins are essential co-activators of the Six family of transcription factors and contain a unique tyrosine phosphatase domain belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for the transcription of a subset of Six1-target genes, and also directs cells to the repair rather than apoptosis pathway upon DNA damage. Furthermore, Eya phosphatase activity has been shown to mediate transformation, invasion, migration, and metastasis of breast cancer cells, making it a potential new drug target for breast cancer. We have previously identified a class of N-arylidenebenzohydrazide compounds that specifically inhibit the Eya2 phosphatase. Herein, we demonstrate that these compounds are reversible inhibitors that selectively inhibit the phosphatase activity of Eya2, but not Eya3. Our mutagenesis results suggest that this class of compounds does not bind to the active site and the binding does not require the coordination with Mg(2+). Moreover, these compounds likely bind within a site on the opposite face of the active site, and function as allosteric inhibitors. We also demonstrate that this class of compounds inhibits Eya2 phosphatase-mediated cell migration, setting the foundation for these molecules to be developed into chemical probes for understanding the specific function of the Eya2 phosphatase and to serve as a prototype for the development of Eya2 phosphatase specific anti-cancer drugs.