Steve Titus

A TRP channel in the lysosome regulates large particle phagocytosis via focal exocytosis.

Phagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. Here, we identified mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca2+ channel regulating focal exocytosis and phagosome biogenesis. Both particle ingestion and lysosomal exocytosis are inhibited by synthetic TRPML1 blockers and are defective in macrophages isolated from TRPML1 knockout mice. Furthermore, TRPML1 overexpression and TRPML1 agonists facilitate both lysosomal exocytosis and particle uptake. Using time-lapse confocal imaging and direct patch clamping of phagosomal membranes, we found that particle binding induces lysosomal PI(3,5)P2 elevation to trigger TRPML1-mediated lysosomal Ca2+ release specifically at the site of uptake, rapidly delivering TRPML1-resident lysosomal membranes to nascent phagosomes via lysosomal exocytosis. Thus phagocytic ingestion of large particles activates a phosphoinositide- and Ca2+-dependent exocytosis pathway to provide membranes necessary for pseudopod extension, leading to clearance of senescent and apoptotic cells in vivo.

Small-molecule agonists for the thyrotropin receptor stimulate thyroid function in human thyrocytes and mice

Seven-transmembrane-spanning receptors (7TMRs) are prominent drug targets. However, small-molecule ligands for 7-transmembrane-spanning receptors for which the natural ligands are large, heterodimeric glycoprotein hormones, like thyroid-stimulating hormone (TSH; thyrotropin), have only recently been reported, and none are approved for human use. We have used quantitative high-throughput screening to identify a small-molecule TSH receptor (TSHR) agonist that was modified to produce a second agonist with increased potency. We show that these agonists are highly selective for human TSHR versus other glycoprotein hormone receptors and interact with the receptors serpentine domain. A binding pocket within the transmembrane domain was defined by docking into a TSHR homology model and was supported by site-directed mutagenesis. In primary cultures of human thyrocytes, both TSH and the agonists increase mRNA levels for thyroglobulin, thyroperoxidase, sodium iodide symporter, and deiodinase type 2, and deiodinase type 2 enzyme activity. Moreover, oral administration of the agonist stimulated thyroid function in mice, resulting in increased serum thyroxine and thyroidal radioiodide uptake. Thus, we discovered a small molecule that activates human TSHR in vitro, is orally active in mice, and could be a lead for development of drugs to use in place of recombinant human TSH in patients with thyroid cancer.

Quantitative High Throughput Screening Using a Live Cell cAMP Assay Identifies Small Molecule Agonists of the TSH Receptor

The thyroid-stimulating hormone (TSH; thyrotropin) receptor belongs to the glycoprotein hormone receptor subfamily of 7-transmembrane spanning receptors. TSH receptor (TSHR) is expressed mainly in thyroid follicular cells and is activated by TSH, which regulates the growth and function of thyroid follicular cells. Recombinant TSH is used in diagnostic screens for thyroid cancer, especially in patients after thyroid cancer surgery. Currently, no selective small-molecule agonists of the TSHR are available. To screen for novel TSHR agonists, the authors miniaturized a commercially available cell-based cyclic adenosine 3′,5′ monophosphate (cAMP) assay into a 1536-well plate format. This assay uses an HEK293 cell line stably transfected with the TSHR coupled to a cyclic nucleotide gated ion channel as a biosensor. From a quantitative high-throughput screen of 73,180 compounds in parallel with a parental cell line (without the TSHR), 276 primary active compounds were identified. The activities of the selected active compounds were further confirmed in an orthogonal homogeneous time-resolved fluorescence cAMP-based assay. Forty-nine compounds in several structural classes have been confirmed as the small-molecule TSHR agonists that will serve as a starting point for chemical optimization and studies of thyroid physiology in health and disease. (Journal of Biomolecular Screening 2008:120-127)

A Small Molecule Inverse Agonist for the Human Thyroid-Stimulating Hormone Receptor

Small molecule inverse agonists for the TSH receptor (TSHR) may be used as probes of the role of basal (or agonist-independent or constitutive) signaling and may have therapeutic potential as orally active drugs to inhibit basal signaling in patients with thyroid cancer and in some patients with hyperthyroidism. We describe the first small-molecule ligand [1;2-(3-((2,6-dimethylphenoxy)methyl)-4-methoxyphenyl)-3-(furan-2-ylmethyl)-2,3-dihydroquinazolin-4(1H)-one] that exhibits inverse agonist properties at TSHR. 1 inhibits basal and TSH-stimulated signaling, measured as cAMP production, by TSHRs in HEK-EM 293 cells stably expressing wild-type TSHRs; the antagonism of TSH-mediated signaling is competitive. 1 also inhibits basal signaling by wild-type TSHRs, and four constitutively active mutants of TSHR expressed transiently in HEK-EM 293 cells. 1 was active under more physiologically relevant conditions in primary cultures of human thyrocytes expressing endogenous TSHRs where it inhibited basal levels of mRNA transcripts for thyroglobulin, thyroperoxidase, sodium iodide symporter, and TSHR. These data serve as proof of principle that small, drug-like molecules can inhibit basal signaling by TSHR. We suggest that this small molecule is a lead compound for the development of higher-potency inverse agonists that can be used as probes of TSHR biology with therapeutic potential.

Discovery and Characterization of a G Protein–Biased Agonist That Inhibits β-Arrestin Recruitment to the D2 Dopamine Receptor

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.

Discovery, Synthesis and Biological Evaluation of Novel SMN Protein Modulators

Spinal muscular atrophy (SMA) is an autosomal recessive disorder affecting the expression or function of survival motor neuron protein (SMN) due to the homozygous deletion or rare point mutations in the survival motor neuron gene 1 (SMN1). The human genome includes a second nearly identical gene called SMN2 that is retained in SMA. SMN2 transcripts undergo alternative splicing with reduced levels of SMN. Up-regulation of SMN2 expression, modification of its splicing, or inhibition of proteolysis of the truncated protein derived from SMN2 have been discussed as potential therapeutic strategies for SMA. In this manuscript, we detail the discovery of a series of arylpiperidines as novel modulators of SMN protein. Systematic hit-to-lead efforts significantly improved potency and efficacy of the series in the primary and orthogonal assays. Structure-property relationships including microsomal stability, cell permeability, and in vivo pharmacokinetics (PK) studies were also investigated. We anticipate that a lead candidate chosen from this series may serve as a useful probe for exploring the therapeutic benefits of SMN protein up-regulation in SMA animal models and a starting point for clinical development.

Comparison on Functional Assays for Gq-Coupled GPCRs by Measuring Inositol Monophospate-1 and Intracellular Calcium in 1536-Well Plate Format

Cell-based functional assays used for compound screening and lead optimization play an important role in drug discovery for G-protein coupled receptors (GPCRs). Cell-based assays can define the role of a compound as an agonist, antagonist or inverse agonist and can provide detailed information about the potency and efficacy of a compound. In addition, cell-based screens can be used to identify allosteric modulators that interact with sites other than the binding site of the endogenous ligand. Intracellular calcium assays which use a fluorescent calcium binding dye (such as Fluo-3, Fluo-4 or Fura-2) have been used in compound screening campaigns to measure the activity of Gq-coupled GPCRs. However, such screening methodologies require a special instrumentation to record the rapid change in intracellular free calcium concentration over time. The radioactive inositol 1,4,5- triphosphate (IP3) assay measures 3H-inositol incorporation and is another traditional assay for the assessment of Gq-coupled GPCR activity, but it is not suitable for screening of large size compound collections because it requires a cell wash step and generates radioactive waste. To avoid these limitations, we have optimized and miniaturized a TR-FRET based IP-One assay that measures inositol monophosphate in a 1536-well plate format. This assay is homogenous, non-radioactive and does not require a kinetic readout. It has been tested with the cell lines expressing M1 acetylcholine, FFAR1, vasopressin V1b, or Neuropeptide S receptors. The activities of antagonists determined in the IP-One assay correlated well with these measured in the intracellular calcium assay while the correlation of agonist activities might vary from cell line to cell line. This IP-One assay offers an alternative method for high throughput screening of Gq-coupled GPCRs without using costly kinetic plate readers.

Tetracycline-Inducible Gene Expression in Conditionally Immortalized Mouse Podocytes

Background: Conditionally immortalized podocytes are valuable research tools but are difficult to efficiently transfect and do not provide graded transgene expression. Methods: Conditionally immortalized mouse podocyte cell lines were established employing a tetracycline-inducible system. Glomerular cells, isolated from transgenic mice bear- ing two transgenes, NPHS2-reverse tetracycline-controlled transactivator, rtTA (A transgene) and H2-Kb-thermosensitive SV40 T, ts58A (I transgene), were cloned. One clone (AI podocytes) expressing WT1 and synaptopodin was transfected with pBI-EGFP (enhanced green fluorescent protein, G transgene) and separately with ptTS-Neo (transcriptional suppressor, T transgene) to produce stable transformants, AIG podocytes and AIT podocytes. Results: AIG podocytes expressed EGFP at 33 and 37°C after doxycycline treatment, and retained podocin and rtTA mRNA expression and temperature-sensitive growth regulation. AIT podocytes, transiently transfected with luciferase-BI-EGFP (LG transgene), showed reduced background expression of EGFP and luciferase in the absence of doxycycline. In AITLG podocytes, generated by stable transfection of AIT podocytes with the LG transgene, luciferase expression was tightly regulated by doxycycline in a time- and concentration-dependent manner both at 33 and 37°C, although background expression was not entirely eliminated. These podocytes retained temperature-sensitive growth regulation and expression of podocyte differentiation markers. Conclusion: Mouse podocytes expressed tetracycline-induced transgenes efficiently while retaining differentiation markers.

Discovery, Optimization, and Characterization of Novel D2 Dopamine Receptor Selective Antagonists

The D2 dopamine receptor (D2 DAR) is one of the most validated drug targets for neuropsychiatric and endocrine disorders. However, clinically approved drugs targeting D2 DAR display poor selectivity between the D2 and other receptors, especially the D3 DAR. This lack of selectivity may lead to undesirable side effects. Here we describe the chemical and pharmacological characterization of a novel D2 DAR antagonist series with excellent D2 versus D1, D3, D4, and D5 receptor selectivity. The final probe 65 was obtained through a quantitative high-throughput screening campaign, followed by medicinal chemistry optimization, to yield a selective molecule with good in vitro physical properties, metabolic stability, and in vivo pharmacokinetics. The optimized molecule may be a useful in vivo probe for studying D2 DAR signal modulation and could also serve as a lead compound for the development of D2 DAR-selective druglike molecules for the treatment of multiple neuropsychiatric and endocrine disorders.

Large-scale pharmacological profiling of 3D tumor models of cancer cells

The discovery of chemotherapeutic agents for the treatment of cancer commonly uses cell proliferation assays in which cells grow as two-dimensional (2D) monolayers. Compounds identified using 2D monolayer assays often fail to advance during clinical development, most likely because these assays do not reproduce the cellular complexity of tumors and their microenvironment in vivo. The use of three-dimensional (3D) cellular systems have been explored as enabling more predictive in vitro tumor models for drug discovery. To date, small-scale screens have demonstrated that pharmacological responses tend to differ between 2D and 3D cancer cell growth models. However, the limited scope of screens using 3D models has not provided a clear delineation of the cellular pathways and processes that differentially regulate cell survival and death in the different in vitro tumor models. Here we sought to further understand the differences in pharmacological responses between cancer tumor cells grown in different conditions by profiling a large collection of 1912 chemotherapeutic agents. We compared pharmacological responses obtained from cells cultured in traditional 2D monolayer conditions with those responses obtained from cells forming spheres versus cells already in 3D spheres. The target annotation of the compound library screened enabled the identification of those key cellular pathways and processes that when modulated by drugs induced cell death in all growth conditions or selectively in the different cell growth models. In addition, we also show that many of the compounds targeting these key cellular functions can be combined to produce synergistic cytotoxic effects, which in many cases differ in the magnitude of their synergism depending on the cellular model and cell type. The results from this work provide a high-throughput screening framework to profile the responses of drugs both as single agents and in pairwise combinations in 3D sphere models of cancer cells.