scholarly journals The Small Molecule Img-98, a Potent and Selective Inhibitor of the Lysine Demethylase Lsd-1, Effectively Augments the Pro-Differentiation Effects of ATRA in a Pre-Clinical Model of AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 460-460 ◽  
Author(s):  
Ronan T. Swords ◽  
Aymee Perez ◽  
Ana Rodriguez ◽  
Justin M. Watts ◽  
Fernando Vargas ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Cell Lines ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Heavy Atom ◽  
Screening Assay ◽  
Inhibitor Screening ◽  
Chemical Complexity ◽  
Lysine Demethylase ◽  
Cayman Chemical

Abstract Acute Promyelocytic Leukemia (APL) is a cytogenetically unique subtype of acute myeloid leukemia (AML), characterized by the presence of the t(15;17)-associated PML-RARA fusion gene. This disease is curable in most patients with all-trans-retinoic acid (ATRA) based therapies, which effectively differentiate malignant promyelocytes. In patients with non-APL AML, most patients with die from their disease and ATRA has little activity. Therefore, research strategies that seek to extend the efficacy of ATRA-based treatment in AML are key avenues of investigation. From our previous studies, an epigenetic analysis of primary AML samples revealed that relative to normal CD33+ cells, loss of RARα2 expression in AML is associated with a reduction in H3K4me2 on the RARA2 promoter (a modification that is associated with transcriptional activation). The mono- and di-methyl lysine demethylase LSD1 (KDM1A) is highly expressed in patients with AML, and its overexpression has been implicated in various other tumors. Based on these data we correctly predicted that the use of small-molecule inhibitors targeting LSD1 (LSD1i) could result in epigenetic reprogramming that enhanced or facilitated the execution of the ATRA-induced differentiation program in AML cells. In the current study, we characterized a range of small molecule inhibitors of LSD-1. All the agents tested (RN-1, GSKi, SP2509, TCP, IMG-98 and OG-L002) led to inhibition of LSD-1 in a biochemical assay with varying degrees of potency. From this study, we further characterized the anti-tumor effects of IMG-98 alone and in combination with ATRA. IMG-98 is a novel LSD1 inhibitor relative to drugs of this class with comparatively different specificity, potency, pharmacokinetics, and metabolism. Its greater heavy atom count and chemical complexity contribute to these properties. By fluorine nuclear magnetic resonance (fNMR) and florescent spectrophotometry, the molecule rapidly reacts irreversibly with the FAD co-factor of LSD1 and this polypeptide is necessary to catalyze the reaction. Thermal stability shifts show the inactivated form of the enzyme becomes much more stable suggesting significant structural changes. Treatment with IMG-98 promoted the expression of the cell surface marker CD11b, associated with a differentiated immunophenotype, in both AML cell lines and primary patient material. IMG-98 produced a potent anti-proliferative effect across a range of AML cell lines and also led to growth inhibition of AML blast colony forming ability. In combination studies with ATRA, IMG-98 re-sensitized AML cells to ATRA by reactivating ATRA driven differentiation programs. Post-differentiation apoptosis was more significant for combined therapy (ATRA + IMG-98) than with either agent alone. Heatmap display of unsupervised hierarchical clustering of genes in AML cell lines differentially expressed in response to treatment with combinations of ATRA, IMG-98 or the combination, confirmed that ATRA combined with IMG-98 enhanced the expression of a subset of genes associated with the myeloid differentiation program. Updated studies on mechanisms underpinning mode of action of IMG-98 in this model will be presented. Taken together, these data demonstrate that ATRA combined with pharmacological inhibition of LSD1, may provide a promising treatment for AML by promoting differentiation and subsequent growth inhibition of AML blasts. A closely related molecule to IMG-98 is currently being optimized in late preclinical development, and clinical trials with this compound are anticipated to start in 2016. Figure 1. Comparative screening assay for LSD1 inhibition with commercially available agents (LSD1 Inhibitor Screening Assay, Cayman Chemical, Cat# 700120) Figure 1. Comparative screening assay for LSD1 inhibition with commercially available agents (LSD1 Inhibitor Screening Assay, Cayman Chemical, Cat# 700120) Disclosures Rienhoff: Imago: Employment.

scholarly journals High-Throughput Screening Assay for Identification of Small Molecule Inhibitors of Aurora2/STK15 Kinase

2004 ◽  
Vol 9 (5) ◽  
pp. 391-397 ◽  
Author(s):  
Chongbo Sun ◽  
Yvette Newbatt ◽  
Leon Douglas ◽  
Paul Workman ◽  
Wynne Aherne ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Tumor Development ◽  
Screening Assay ◽  
Amino Terminal ◽  
Terminal Domain ◽  
High Throughput Screening Assay ◽  
Amino Terminal Domain

STK15/Aurora2 is a centrosome-associated serine/threonine kinase, the protein levels and kinase activity of which rise during G2 and mitosis. STK15 overexpression induces tumorigenesis and is amplified in various human cancers and tumor cell lines. Thus, STK15 represents an important therapeutic target for small molecule inhibitors that would disrupt its activity and block cell proliferation. The availability of a robust and selective small molecule inhibitor would also provide a useful tool for identification of the potential role of STK15 in cell cycle regulation and tumor development. The authors report the development of a novel, fast, simple microplate assay for STK15 activity suitable for high-throughput screening. In the assay, γ-33P-ATP and STK15 were incubated in a myelin basic protein (MBP)-coated FlashPlate® to generate a scintillation signal. The assay was reproducible, the signal-to-noise ratio was high (11) and the Z′ factor was 0.69. The assay was easily adapted to a robotic system for drug discovery programs targeting STK15. The authors also demonstrate that STK15 is regulated by phosphorylation and the N-amino terminal domain of the protein. Treatment with phosphatase inhibitors (okadaic acid) or deletion of the N-amino terminal domain results in a significant increase in the enzymatic activity.

scholarly journals A chemoprobe tracks its target

2019 ◽  
Vol 294 (20) ◽  
pp. 8323-8324
Author(s):  
Aseem Z. Ansari
Keyword(s):  
Small Molecule ◽  
Clinical Utility ◽  
Small Molecule Inhibitors ◽  
Indirect Measure ◽  
Histone Marks ◽  
Histone Lysine ◽  
Histone Lysine Demethylase ◽  
Histone Modifying Enzymes ◽  
Lysine Demethylase ◽  
In Cells

Small-molecule inhibitors of histone-modifying enzymes have significant clinical utility for managing diseases such as cancer. These inhibitors are usually identified and monitored through their effects on the gain or loss of specific histone marks. In cells, multiple related enzymes can place or remove a specific mark; therefore, relying on an indirect measure of inhibitor engagement can be misleading. Mascaró et al. describe a luminescence-based ELISA approach that directly monitors binding of inhibitors to the histone lysine demethylase KDM1A.

scholarly journals A High-Throughput Cellular Screening Assay for Small-Molecule Inhibitors and Activators of Cytoplasmic Dynein-1-Based Cargo Transport

2020 ◽  
Vol 25 (9) ◽  
pp. 985-999
Author(s):  
John Vincent ◽  
Marian Preston ◽  
Elizabeth Mouchet ◽  
Nicolas Laugier ◽  
Adam Corrigan ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Cytoplasmic Dynein ◽  
Lead Discovery ◽  
Screening Assay ◽  
Age Related ◽  
The Uk

Cytoplasmic dynein-1 (hereafter dynein) is a six-subunit motor complex that transports a variety of cellular components and pathogens along microtubules. Dynein’s cellular functions are only partially understood, and potent and specific small-molecule inhibitors and activators of this motor would be valuable for addressing this issue. It has also been hypothesized that an inhibitor of dynein-based transport could be used in antiviral or antimitotic therapy, whereas an activator could alleviate age-related neurodegenerative diseases by enhancing microtubule-based transport in axons. Here, we present the first high-throughput screening (HTS) assay capable of identifying both activators and inhibitors of dynein-based transport. This project is also the first collaborative screening report from the Medical Research Council and AstraZeneca agreement to form the UK Centre for Lead Discovery. A cellular imaging assay was used, involving chemically controlled recruitment of activated dynein complexes to peroxisomes. Such a system has the potential to identify molecules that affect multiple aspects of dynein biology in vivo. Following optimization of key parameters, the assay was developed in a 384-well format with semiautomated liquid handling and image acquisition. Testing of more than 500,000 compounds identified both inhibitors and activators of dynein-based transport in multiple chemical series. Additional analysis indicated that many of the identified compounds do not affect the integrity of the microtubule cytoskeleton and are therefore candidates to directly target the transport machinery.

Multidrug Resistance Reversal Agent, NSC77037, Identified with a Cell-Based Screening Assay

2010 ◽  
Vol 15 (3) ◽  
pp. 287-296 ◽  
Author(s):  
Michiro Susa ◽  
Edwin Choy ◽  
Cao Yang ◽  
Joseph Schwab ◽  
Henry Mankin ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Cancer Patients ◽  
Cell Lines ◽  
Cancer Cell ◽  
Small Molecule ◽  
Cancer Resistance ◽  
Screening Assay ◽  
A Cell ◽  
Resistance Protein ◽  
Mdr Reversal

The development of multidrug resistance (MDR) remains a significant obstacle in treating cancer patients with chemotherapy. To identify small-molecule compounds that can reverse MDR, the authors used a cell-based screening assay with an MDR ovarian cancer cell line. Incubating MDR cells with a sublethal concentration of paclitaxel in combination with each of 2000 small-molecule compounds from the National Cancer Institute Diversity Set Library, they identified NSC77037. The cytotoxic activity of NSC77037 and the duration of its effect were evaluated in vitro using a panel of cancer cell lines expressing permeability glycoprotein (Pgp), multiple drug resistance protein 1 (MRP 1), and breast cancer resistance protein (BCRP). The mechanism of its effects was further analyzed by assessing the retention of calcein and Pgp-ATPase activity. The relative potency of MDR reversal by NSC77037 was significantly higher than that of frequently used MDR reversal agents such as verapamil and cyclosporine A. NSC77037 reversed Pgp without reversing MRP or BCRP-mediated MDR. NSC77037, at a concentration of >10 µM, moderately inhibited the proliferation of both sensitive and resistant cell lines, but the inhibitory effect of NSC77037 was not altered by coincubation with the Pgp inhibitor verapamil, suggesting that NSC77037 itself is not a substrate of Pgp. NSC77037 directly inhibited the function of Pgp in a dose-dependent manner, but it did not alter the protein expression level of Pgp. The use of NSC77037 to restore sensitivity to chemotherapy or to prevent resistance could be a potential treatment strategy for cancer patients.

scholarly journals Protein kinase Cδ inactivation inhibits cellular proliferation and decreases survival in human neuroendocrine tumors

2011 ◽  
Vol 18 (6) ◽  
pp. 759-771 ◽  
Author(s):  
Zhihong Chen ◽  
Lora W Forman ◽  
Kenneth A Miller ◽  
Brandon English ◽  
Asami Takashima ◽  
...  
Keyword(s):  
Cell Lines ◽  
Neuroendocrine Tumors ◽  
Small Molecule ◽  
Cellular Proliferation ◽  
Small Molecule Inhibitors ◽  
Cancer Therapeutics ◽  
Therapeutic Modality ◽  
Ras Signaling ◽  
Normal Tissues ◽  
Induced Apoptosis

The concept of targeting cancer therapeutics toward specific mutations or abnormalities in tumor cells, which are not found in normal tissues, has the potential advantages of high selectivity for the tumor and correspondingly low secondary toxicities. Many human malignancies display activating mutations in the Ras family of signal-transducing genes or over-activity of p21Ras-signaling pathways. Carcinoid and other neuroendocrine tumors have been similarly demonstrated to have activation of Ras signaling directly by mutations in Ras, indirectly by loss of Ras-regulatory proteins, or via constitutive activation of upstream or downstream effector pathways of Ras, such as growth factor receptors or PI3-kinase and Raf/mitogen-activated protein kinases. We previously reported that aberrant activation of Ras signaling sensitizes cells to apoptosis when the activity of the PKCδ isozyme is suppressed and that PKCδ suppression is not toxic to cells with normal levels of p21Rassignaling. We demonstrate here that inhibition of PKCδ by a number of independent means, including genetic mechanisms (shRNA) or small-molecule inhibitors, is able to efficiently and selectively repress the growth of human neuroendocrine cell lines derived from bronchopulmonary, foregut, or hindgut tumors. PKCδ inhibition in these tumors also efficiently induced apoptosis. Exposure to small-molecule inhibitors of PKCδ over a period of 24 h is sufficient to significantly suppress cell growth and clonogenic capacity of these tumor cell lines. Neuroendocrine tumors are typically refractory to conventional therapeutic approaches. This Ras-targeted therapeutic approach, mediated through PKCδ suppression, which selectively takes advantage of the very oncogenic mutations that contribute to the malignancy of the tumor, may hold potential as a novel therapeutic modality.

scholarly journals Methionine Restriction As a Novel Therapeutic Strategy for MLL (KMT2A)-Rearranged Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2269-2269
Author(s):  
Trisha Tee ◽  
Titine Ruiter ◽  
Ahmed Dahaoui ◽  
Dorette van Ingen Schenau ◽  
Rico Hagelaar ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Patient Group ◽  
Small Molecule ◽  
Metabolic Activity ◽  
Small Molecule Inhibitors ◽  
Lymphoblastic Leukemia ◽  
Childhood All ◽  
Methionine Restriction ◽  
Novel Therapeutic

Abstract Background: MLL (KMT2A)-rearranged acute lymphoblastic leukemia (MLLr ALL) is a rare but aggressive subset that represents 5% of childhood ALL cases, and accounts for about 70% of infant leukemias. While overall survival in these young children is around 50%, after relapse, MLLr ALL becomes an almost incurable disease, highlighting the urgent clinical need for new strategies for this patient group. The histone methyl transferase function of the MLL fusion protein complex requires the methionine metabolite s-adenosylmethionine (SAM) as methyl donor, suggesting a selective sensitivity of MLL-r ALL for perturbations in methionine availability. Recent studies in solid tumor models suggest clinical utility of methionine restricted diets or oral administration of methionine depleting enzyme Methionine Gamma Lyase (MGL) to be safe and effective. Therefore, we explored the effect of methionine restriction (MR) as a potential, new therapy for MLLr ALL. Methods: We compared the effects of MR on metabolic activity and viability between MLLr and non-MLLr pre-BCP ALL cell lines using enzymatic depletion, small molecule inhibitors targeting methionine metabolism, and restrictive culture conditions. To identify intrinsic metabolic differences between MLLr and non-MLLr cells and explore how MR impinges on their metabolic state, we performed global metabolomics on MLLr SEM cells and non-MLLr NALM6 cells cultured with complete depletion of methionine. Additionally, we used RNA sequencing to assess the global effects of MR on gene expression, and a CRISPR/Cas9-based reverse genetic screen to identify sensitizers towards MR. Results were validated in vitro using targeted knockouts and small-molecule inhibitors, as well as in vivo using a 95% methionine restricted diet. Immunocompromised mice were engrafted with MLLr SEM cells and 7 days after transplantation, mice were randomized to control or 95% MR diet. Leukemia progression was monitored by flowcytometric detection of human lymphocytes in the blood. Results: We observed that depletion of methionine reduces metabolic activity in almost all BCP-ALL (B-ALL) cell lines, however, only in MLLr B-ALL cell lines was rapid apoptosis induced (Figure 1A). Global metabolic profiling revealed significant basal metabolic differences, of note being SAM, whose levels were approximately 5-fold higher in MLLr SEM cells compared to non-MLLr NALM6 cells. Consistent with this, addition of SAM completely rescued MLLr cell lines from methionine depletion induced apoptosis, an effect not observed in non-MLLr cells (Figure 1A). Metabolomic profiling also highlighted different salvage mechanisms at play in NALM6 cells, with the folate cycle and polyamine synthesis pathway being activated upon MR. Together, these results indicate that MLLr B-ALL cells are selectively sensitive to MR. In line with this, RNASeq data showed significant decreased expression of several known MLL fusion target genes such as PROM1, HOXA10, and MEIS1 in response to MR. To obtain further insight into the pathways involved in the response to MR and to identify potential therapeutic targets that further sensitize cells to MR, we performed a CRISPR/Cas9-based screen. This identified three members of the Bromodomain- and extra-terminal domain (BET) family as potential modifiers of the response to MR in SEM cells. Indeed, RNAseq analysis showed that Myc activity as a proxy of BRD4 function, was strongly suppressed by MR. Finally, preliminary results show the efficacy of dietary intervention alone on leukemia progression. We observe with 95% MR diet, significant delays on leukemic growth (Figure 1B). Moreover, the MR diet was well tolerated, as indicated by minimal weight loss after two months. Although further studies are needed, we anticipate that targeting epigenetic regulators or use of conventional therapies in combination with MR would further potentiate this effect. Conclusions: MLLr leukemic cells have an increased dependency on S-adenosylmethionine and therefore show increased vulnerability to methionine depletion. Limiting methionine availability, either by enzymatic methionine depletion or dietary restriction could provide a novel therapeutic option for this patient group, particularly when combined with other therapies. The availability of an FDA approved methionine-free formula facilitates rapid translation to clinical practice, particularly in infants. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Ranking small molecules by how much they preferentially inhibit the growth of cancer cell lines with either BRAF or KRAS oncogene mutations

2014 ◽  
Author(s):  
James Langham
Keyword(s):  
Growth Inhibition ◽  
Small Molecules ◽  
Cell Line ◽  
Cell Lines ◽  
Small Molecule ◽  
Molecular Mechanisms ◽  
Braf Inhibitor ◽  
Data Set ◽  
Cytidine Analogs ◽  
Knowledge Of Cancer

We were interested in the question of whether it might be possible to use knowledge of cancer-related mutations in the cell lines of the NCI60 screening data set to identify small molecules that preferentially inhibit the growth of cell lines containing either BRAF or KRAS oncogene mutations. Our hypothesis was that this cell line mutation knowledge could help to identify small molecules that were more likely to preferentially inhibit growth of cell lines with a particular mutation. It seems that any such molecules might be further investigated to try to better understand the molecular mechanisms of growth inhibition. We defined a quantity, \(\text{Diff}_{\text{mut}}\), that estimates how much more a given small molecule inhibits cell lines with a mutation of interest than cell lines without that mutation. We ranked the small molecules in descending order of \(\text{Diff}_{\text{mut}}\) and then tried to explain whether the ranking of the highest ranked molecules made sense in terms of independent facts about these molecules. This method showed the BRAF inhibitor vemurafenib to be highly ranked in the BRAF ranking. The cytidine analog cytarabine was found to be highly ranked in the KRAS ranking. Other cytidine analogs were also found to be highly ranked with respect to KRAS.

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