Identification of Novel GANT61 Analogs with Activity in Hedgehog Functional Assays and GLI1-Dependent Cancer Cells.

Aberrant activation of hedgehog (Hh) signaling has been implicated in various cancers. Current FDA-approved inhibitors target the seven-transmembrane receptor Smoothened, but resistance to these drugs has been observed. It has been proposed that a more promising strategy to target this pathway is at the GLI1 transcription factor level. GANT61 was the first small molecule identified to directly suppress GLI-mediated activity; however, its development as a potential anti-cancer agent has been hindered by its modest activity and aqueous chemical instability. Our study aimed to identify novel GLI1 inhibitors. JChem searches identified fifty-two compounds similar to GANT61 and its active metabolite, GANT61-D. We combined high-throughput cell-based assays and molecular docking to evaluate these analogs. Five of the fifty-two GANT61 analogs inhibited activity in Hh-responsive C3H10T1/2 and Gli-reporter NIH3T3 cellular assays without cytotoxicity. Two of the GANT61 analogs, BAS 07019774 and Z27610715, reduced Gli1 mRNA expression in C3H10T1/2 cells. Treatment with BAS 07019774 significantly reduced cell viability in Hh-dependent glioblastoma and lung cancer cell lines. Molecular docking indicated that BAS 07019774 is predicted to bind to the ZF4 region of GLI1, potentially interfering with its ability to bind DNA. Our findings show promise in developing more effective and potent GLI inhibitors.

Discovery of small molecule inhibitors for the C. elegans caspase CED-3 by high-throughput screening.

C. elegans has been widely used as a model organism for programmed cell death and apoptosis. Although the CED-3 caspase is the primary effector of cell death in C. elegans, no selective inhibitors have been identified. Utilizing high-throughput screening with recombinant C. elegans CED-3 protein, we have discovered and confirmed 21 novel small molecule inhibitors. Six compounds had IC50 values < 10 µM. From these, four distinct chemotypes were identified. The inhibitor scaffolds described here could lead to the development of selective molecular probes to facilitate our understanding of programmed cell death in this model organism.

Identification of a DYRK1A-mediated phosphorylation site within the nuclear localization sequence of the hedgehog transcription factor GLI1.

GLI1 is a key downstream transcription effector of the Hedgehog (Hh) signaling pathway that is involved in promoting cell growth, differentiation and tissue patterning in embryonic development. GLI1 over-activation and its nuclear localization has also been linked to the increased aggressiveness of a number of cancers. It has previously been demonstrated that DYRK1A (dual-specificity tyrosine-regulated kinase 1A) can phosphorylate GLI1 and promote GLI1 nuclear localization and its transcriptional activity. Utilizing recombinant human GLI1 and DYRK1A proteins and phospho-peptide mass spectrometry, we demonstrated that GLI1 is phosphorylated by DYRK1A at Ser408, a phospho-site that falls within the putative nuclear localization sequence (NLS) of GLI1, suggesting a possible mechanistic role in modulating its translocation. Further, we showed that the Ser408 site on GLI1 was not phosphorylated in the presence of the selective DYRK1A inhibitor harmine. The data described herein provide the first identification of a DYRK1A-mediated site of phosphorylation on GLI1 within its NLS and may serve as a valuable mechanism for further understanding Hh signaling modulation.

Recombinant human sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) is expressed at high yield as an active homotetramer in baculovirus-infected insect cells.

The sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) isoform is a promising contraceptive target because it is specific to male germ cells, essential for sperm motility and male fertility, and well suited to pharmacological inhibition. However, GAPDHS is difficult to isolate from native sources and recombinant expression frequently results in high production of insoluble enzyme. We chose to use the Bac-to-Bac baculovirus-insect cell system to express a His-tagged form of human GAPDHS (Hu his-GAPDHS) lacking the proline-rich N-terminal sequence. This recombinant Hu his-GAPDHS was successfully produced in Spodoptera frugiperda 9 (Sf9) cells by infection with recombinant virus as a soluble, enzymatically active form in high yield, >35 mg/L culture. Biochemical characterization of the purified enzyme by mass spectrometry and size exclusion chromatography confirmed the presence of the tetrameric form. Further characterization by peptide ion matching mass spectrometry and Edman sequencing showed that unlike the mixed tetramer forms produced in bacterial expression systems, human his-GAPDHS expressed in baculovirus-infected insect cells is homotetrameric. The ability to express and purify active human GAPDHS as homotetramers in high amounts will greatly aid in drug discovery efforts targeting this enzyme for discovery of novel contraceptives and three compounds were identified as inhibitors of Hu his-GAPDHS from a pilot screen of 1120 FDA-approved compounds.

Development and validation of a hedgehog heparin-binding assay for high-throughput screening.

Sonic hedgehog (Shh) is a morphogenic protein with critical roles in embryogenesis and the development of some cancers. Hence, identifying inhibitors of the Shh pathway is of great therapeutic value. Heparin and HSPGs act as crucial modulators of Shh activity. To identify molecules that antagonize Shh binding to heparin we have developed a solid-phase plate-based assay. The N-terminal domain of Shh (ShhN) protein is first coated in 384-well plates and the direct binding of fluorescein-labeled heparin (flu-heparin) assessed by measuring the fluorescence signal after incubation and wash steps. Binding of ShhN protein to the 384-well plates was confirmed and optimized by a standard ELISA using a monoclonal antibody recognizing folded ShhN. The assay was validated using whole plate minimum and maximum signal wells with a Z' of 0.68-0.75 determined. Herein, we describe the development and validation of a high throughput screen to identify small molecule antagonists of Shh heparin binding. Overall, this method•Results in an optimized and validated assay for hedgehog heparin binding.•Delivers a cost effective high-throughput screen format for hedgehog heparin antagonist screening.

Data on peptides identified by mass spectrometry analysis of in vitro DYRK1A-mediated phosphorylation sites on GLI1.

The data presented in this article support the accompanying research article "Identification of a DYRK1A-mediated phosphorylation site within the nuclear localization sequence of the hedgehog transcription factor GLI1" (Ehe et al., 2017) [1]. Although it has been demonstrated that DYRK1A (dual-specificity tyrosine-regulated kinase 1A) can phosphorylate the hedgehog pathway transcription factor GLI1 (GLIoma-associated oncogene homolog 1) and promote its nuclear localization, the DYRK1A-mediated sites of phosphorylation on GLI1 involved were not fully known. This article details the mass spectrometry methods and resulting dataset for the peptides identified from GLI1 when incubated with DYRK1A under varying conditions. The data include details of sequence coverage and all phospho-peptides identified.

Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS).

Glycolytic isozymes that are restricted to the male germline are potential targets for the development of reversible, non-hormonal male contraceptives. GAPDHS, the sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase, is an essential enzyme for glycolysis making it an attractive target for rational drug design. Toward this goal, we have optimized and validated a high-throughput spectrophotometric assay for GAPDHS in 384-well format. The assay was stable over time and tolerant to DMSO. Whole plate validation experiments yielded Z' values >0.8 indicating a robust assay for HTS. Two compounds were identified and confirmed from a test screen of the Prestwick collection. This assay was used to screen a diverse chemical library and identified fourteen small molecules that modulated the activity of recombinant purified GAPDHS with confirmed IC50 values ranging from 1.8 to 42 µM. These compounds may provide useful scaffolds as molecular tools to probe the role of GAPDHS in sperm motility and long term to develop potent and selective GAPDHS inhibitors leading to novel contraceptive agents.