Emma Fairweather

Development and evaluation of selective, reversible LSD1 inhibitors derived from fragments

Two series of aminothiazoles have been developed as reversible inhibitors of lysine specific demethylase 1 (LSD1) through the expansion of a hit derived from a high concentration biochemical fragment based screen of 2466 compounds. The potency of the initial fragment hit was increased 32-fold through synthesis, with one series of compounds showing clear structure–activity relationships and inhibitory activities in the range of 7 to 187 μM in a biochemical assay. This series also showed selectivity against the related FAD-dependent enzyme mono-amine oxidase A (MAO-A). Although a wide range of irreversible inhibitors of LSD1 have been reported with activities in the low nanomolar range, this work represents one of the first reported examples of a reversible small molecule inhibitor of LSD1 with clear SAR and selectivity against MAO-A, and could provide a platform for the development of more potent reversible inhibitors. Herein, we also report the use of a recently developed cell-based assay for profiling LSD1 inhibitors, and present results on our own compounds as well as a selection of recently described reversible LSD1 inhibitors.

Novel Steroid Inhibitors of Glucose 6-Phosphate Dehydrogenase

Novel derivatives of the steroid DHEA 1, a known uncompetitive inhibitor of G6PD, were designed, synthesized, and tested for their ability to inhibit this dehydrogenase enzyme. Several compounds with approximately 10-fold improved potency in an enzyme assay were identified, and this improved activity translated to efficacy in a cellular assay. The SAR for steroid inhibition of G6PD has been substantially developed; the 3β-alcohol can be replaced with 3β-H-bond donors such as sulfamide, sulfonamide, urea, and carbamate. Improved potency was achieved by replacing the androstane nucleus with a pregnane nucleus, provided a ketone at C-20 is present. For pregnan-20-ones incorporation of a 21-hydroxyl group is often beneficial. The novel compounds generally have good physicochemical properties and satisfactory in vitro DMPK parameters. These derivatives may be useful for examining the role of G6PD inhibition in cells and will assist the future design of more potent steroid inhibitors with potential therapeutic utility.

The discovery of 2-substituted phenol quinazolines as potent RET kinase inhibitors with improved KDR selectivity.

Deregulation of the receptor tyrosine kinase RET has been implicated in medullary thyroid cancer, a small percentage of lung adenocarcinomas, endocrine-resistant breast cancer and pancreatic cancer. There are several clinically approved multi-kinase inhibitors that target RET as a secondary pharmacology but additional activities, most notably inhibition of KDR, lead to dose-limiting toxicities. There is, therefore, a clinical need for more specific RET kinase inhibitors. Herein we report our efforts towards identifying a potent and selective RET inhibitor using vandetanib 1 as the starting point for structure-based drug design. Phenolic anilinoquinazolines exemplified by 6 showed improved affinities towards RET but, unsurprisingly, suffered from high metabolic clearance. Efforts to mitigate the metabolic liability of the phenol led to the discovery that a flanking substituent not only improved the hepatocyte stability, but could also impart a significant gain in selectivity. This culminated in the identification of 36; a potent RET inhibitor with much improved selectivity against KDR.

Discovery and Optimization of Allosteric Inhibitors of Mutant Isocitrate Dehydrogenase 1 (R132H IDH1) Displaying Activity in Human Acute Myeloid Leukemia Cells

A collaborative high throughput screen of 1.35 million compounds against mutant (R132H) isocitrate dehydrogenase IDH1 led to the identification of a novel series of inhibitors. Elucidation of the bound ligand crystal structure showed that the inhibitors exhibited a novel binding mode in a previously identified allosteric site of IDH1 (R132H). This information guided the optimization of the series yielding submicromolar enzyme inhibitors with promising cellular activity. Encouragingly, one compound from this series was found to induce myeloid differentiation in primary human IDH1 R132H AML cells in vitro.

An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells.

After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.

Anilinoquinazoline inhibitors of the RET kinase domain-Elaboration of the 7-position

Graphical abstract

Enhancer activation by pharmacologic displacement of LSD1 from GFI1 induces differentiation in acute myeloid leukemia

Summary Pharmacologic inhibition of LSD1 promotes blast cell differentiation in acute myeloid leukemia (AML) with MLL translocations. The assumption has been that differentiation is induced through blockade of LSD1’s histone demethylase activity. However, we observed that rapid, extensive, drug-induced changes in transcription occurred without genome-wide accumulation of the histone modifications targeted for demethylation by LSD1 at sites of LSD1 binding and that a demethylase-defective mutant rescued LSD1 knockdown AML cells as efficiently as wild-type protein. Rather, LSD1 inhibitors disrupt the interaction of LSD1 and RCOR1 with the SNAG-domain transcription repressor GFI1, which is bound to a discrete set of enhancers located close to transcription factor genes that regulate myeloid differentiation. Physical separation of LSD1/RCOR1 from GFI1 is required for drug-induced differentiation. The consequent inactivation of GFI1 leads to increased enhancer histone acetylation within hours, which directly correlates with the upregulation of nearby subordinate genes.

Abstract C39: First-in-class inhibitors of the putatively undruggable DNA repair target Poly(ADP-ribose) glycohydrolase (PARG)

Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose) polymerases (PARPs). PARG depletion, using RNAi, results in several effects such as PAR chain persistence, progression of single- to double-strand DNA lesions and NAD+ depletion. Given these findings, inhibition of PARG with a small molecule agent offers a potential opportunity to interfere with DNA repair mechanisms and induce cell death in those cells with increased susceptibility to DNA damage, such as tumour cells. Previous efforts to develop small molecule inhibitors of PARG activity have generally been hampered by poor physicochemical properties, off-target pharmacology and a lack of cell permeability, leading some to suggest that PARG may be undruggable. In contrast, we have now developed a series of first-in-class PARG inhibitors which display drug-like properties and attractive pharmacokinetic parameters. These compounds have proved to be useful biological tool compounds. Moreover, displaying selective activity in both biochemical and, more importantly, cellular assays of PARG function, these derivatives have allowed an exploration of the phenotypes resulting from reversible, pharmacological PARG inhibition in both in vitro cell panels and in vivo models. Furthermore, our initial bioinformatic analysis suggests that deficiency of a known tumour suppressor confers sensitivity to PARG inhibition, suggesting patient populations that will potentially benefit from PARGi therapies. Citation Format: Bohdan Waszkowycz, Dominic James, Ben Acton, Emma Fairweather, Sam Fritzl, Niall Hamilton, Nicola Hamilton, Sarah Holt, James Hitchen, Colin Hutton, Stuart Jones, Allan Jordan, Alison McGonagle, Daniel Mould, Helen Small, Kate Smith, Alexandra Stowell, Ian D. Waddell, Donald Ogilvie. First-in-class inhibitors of the putatively undruggable DNA repair target Poly(ADP-ribose) glycohydrolase (PARG). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C39.

Abstract 5429: Inhibition of SMARCA2: a novel target for SMARCA4-deficient lung adenocarcinoma

Aim: With the decreasing costs of genomics technologies, ever more data is being put into the public domain. Scientific papers only highlight a fraction of the information in the data, consequently further mining can answer drug discovery relevant questions and identify new targets. In this work we developed a bioinformatics pipeline, based on the collateral vulnerability hypothesis, to integrate several sources of public data and identify novel targets to form the basis of a new drug discovery project. Methods: Genomic data from TCGA was integrated with phenotypic data extracted from Mousemine, Flymine and Wormbase to identify loss-of-function aberrations in genes from families with essential predicted function. Follow-up experiments investigated the effect of siRNA knockdown of paralogs of genes of interest on various cellular phenotypes including proliferation, survival and senescence in gene deficient cell lines. A fragment screen was used to assess drugability of genes of interest. Results: The pipeline has been applied to several cancer types, and as a result a drug discovery project has been initiated against SMARCA2 in SMARCA4-deficient lung adenocarcinoma. SMARCA4 is a bromodomain-containing transcriptional co-activator within the multi-subunit SWF/SNF complex, which also possesses helicase and ATPase activities and functions to alter chromatin structure. SMARCA4-deficient cell lines harbour abrogating mutations, and previous studies have demonstrated that knockdown of SMARCA2, its functional paralog, in SMARCA4-deficient cell lines results in reduced cellular proliferation and survival. Moreover, SMARCA2 has been shown to be inactivated by epigenetic silencing in a proportion of human tumours. The collateral vulnerability hypothesis was tested in a panel of lung adenocarcinoma cell lines with SMARCA2- and/or SMARCA4-deficiencies. Experiments investigating the effect of siRNA knockdown confirmed both our hypothesis and the published data. A fragment screen against the bromodomain of SMARCA2 generated a high ‘ligandability’ index, suggesting that this target is druggable. Conclusion: SMARCA2 has been validated by our work and others as a target in SMARCA4 deficient lung adenocarcinoma. Future work will focus on elucidating the role of the bromodomain and the ATPase domain in SMARCA2/4 activity, and we are actively pursuing the identification of small molecule inhibitors of SMARCA2. An HTS has been undertaken against a library of >700 million compounds in a DNA-encoded library to identify novel hit matter that may ultimately be developed for therapeutic value. Citation Format: Phil Chapman, Nikki March, Graeme Thomson, Emma Fairweather, Samantha Fritzl, James Hitchin, Nicola Hamilton, Allan Jordan, Ian Waddell, Donald Ogilvie. Inhibition of SMARCA2: a novel target for SMARCA4-deficient lung adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5429. doi:10.1158/1538-7445.AM2015-5429

Abstract B98: Development and evaluation of selective, reversible LSD1 inhibitors from fragment startpoints.

There is currently considerable interest in lysine-specific histone demethylase 1 (LSD1) as a therapeutic target in human malignancies. Specifically LSD1 has been demonstrated to be an essential regulator of leukaemia stem cell potential, inhibiting differentiation and apoptosis in the MLL-AML setting. There are a variety of potent irreversible LSD1 inhibitors available but here we present two series of reversible aminothiazole inhibitors obtained through the expansion of hits derived from a high concentration biochemical screen of a fragment library. The potency of the initial fragment hits was increased 32-fold through synthesis, with one series of compounds showing clear structure activity relationships (SAR) and inhibitory activities in the range of 7 to 187 µM in a biochemical assay. This series also showed selectivity against the homologous amine oxidase enzyme monoamine oxidase A (MAO-A). This work represents one of the first reported examples of a reversible small molecule inhibitor of LSD1 with clear SAR and selectivity against MAO-A, and could provide a platform for the development of more potent reversible inhibitors. We also report the first Proof of Mechanism (POM) cell based assay utilizing CD86 expression as a surrogate marker of LSD1 activity in THP1 cells and its use to evaluate both our compounds and some recently reported reversible LSD1 inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B98. Citation Format: Alex Stowell, Niall Hamilton, James Hitchin, Julian Blagg, Rosemary Burke, Samantha Burns, Mark J. Cockerill, Emma Fairweather, Colin Hutton, Allan Jordan, Daniel Mould, Graeme Thomson, Ian Waddell, Donald Ogilvie. Development and evaluation of selective, reversible LSD1 inhibitors from fragment startpoints. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B98.