Graeme Thomson

Toxoflavins and Deazaflavins as the First Reported Selective Small Molecule Inhibitors of Tyrosyl-DNA Phosphodiesterase II

The recently discovered enzyme tyrosyl-DNA phosphodiesterase 2 (TDP2) has been implicated in the topoisomerase-mediated repair of DNA damage. In the clinical setting, it has been hypothesized that TDP2 may mediate drug resistance to topoisomerase II (topo II) inhibition by etoposide. Therefore, selective pharmacological inhibition of TDP2 is proposed as a novel approach to overcome intrinsic or acquired resistance to topo II-targeted drug therapy. Following a high-throughput screening (HTS) campaign, toxoflavins and deazaflavins were identified as the first reported sub-micromolar and selective inhibitors of this enzyme. Toxoflavin derivatives appeared to exhibit a clear structure-activity relationship (SAR) for TDP2 enzymatic inhibition. However, we observed a key redox liability of this series, and this, alongside early in vitro drug metabolism and pharmacokinetics (DMPK) issues, precluded further exploration. The deazaflavins were developed from a singleton HTS hit. This series showed distinct SAR and did not display redox activity; however low cell permeability proved to be a challenge.

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.

High Throughput Screens Yield Small Molecule Inhibitors of Leishmania CRK3:CYC6 Cyclin-Dependent Kinase

Background Leishmania species are parasitic protozoa that have a tightly controlled cell cycle, regulated by cyclin-dependent kinases (CDKs). Cdc2-related kinase 3 (CRK3), an essential CDK in Leishmania and functional orthologue of human CDK1, can form an active protein kinase complex with Leishmania cyclins CYCA and CYC6. Here we describe the identification and synthesis of specific small molecule inhibitors of bacterially expressed Leishmania CRK3:CYC6 using a high throughput screening assay and iterative chemistry. We also describe the biological activity of the molecules against Leishmania parasites. Methodology/Principal Findings In order to obtain an active Leishmania CRK3:CYC6 protein kinase complex, we developed a co-expression and co-purification system for Leishmania CRK3 and CYC6 proteins. This active enzyme was used in a high throughput screening (HTS) platform, utilising an IMAP fluorescence polarisation assay. We carried out two chemical library screens and identified specific inhibitors of CRK3:CYC6 that were inactive against the human cyclin-dependent kinase CDK2:CycA. Subsequently, the best inhibitors were tested against 11 other mammalian protein kinases. Twelve of the most potent hits had an azapurine core with structure activity relationship (SAR) analysis identifying the functional groups on the 2 and 9 positions as essential for CRK3:CYC6 inhibition and specificity against CDK2:CycA. Iterative chemistry allowed synthesis of a number of azapurine derivatives with one, compound 17, demonstrating anti-parasitic activity against both promastigote and amastigote forms of L. major. Following the second HTS, 11 compounds with a thiazole core (active towards CRK3:CYC6 and inactive against CDK2:CycA) were tested. Ten of these hits demonstrated anti-parasitic activity against promastigote L. major. Conclusions/Significance The pharmacophores identified from the high throughput screens, and the derivatives synthesised, selectively target the parasite enzyme and represent compounds for future hit-to-lead synthesis programs to develop therapeutics against Leishmania species. Challenges remain in identifying specific CDK inhibitors with both target selectivity and potency against the parasite.

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.

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.

Abstract 3324: The discovery and optimisation of small-molecule inhibitors of human 5’-tyrosyl DNA phosphodiesterase (Tdp2).

Topoisomerases (topo) regulate DNA topology by the transient cleavage and re-ligation of DNA during transcription and replication. Topo II poisons such as etoposide can induce abortive DNA strand breaks in which topo II remains covalently bound to a 5? DNA strand terminus via a phosphotyrosyl linker. Tyrosyl DNA phosphodiesterase 2 (Tdp2, TTRAP, EAPII) is a recently discovered human 5?-tyrosyl DNA phosphodiesterase which repairs this topo-mediated DNA damage, therefore playing a central role in maintaining normal DNA topology in cells. Cellular depletion of Tdp2 has been shown to result in an increased susceptibility and sensitivity to topo II-induced DNA double strand breaks. It has therefore been proposed that selective pharmacological inhibition of Tdp2 may be a novel approach to overcome intrinsic or acquired resistance to topo II targeted drug therapy. To date, no known drug-like inhibitors of Tdp2 have been identified. We have recently reported a robust ?mix and read? HTS compatible assay and this was used to screen a diverse chemical library of approximately 92,000 compounds. From this, 2 distinct hit series have been identified. Following further chemical exploration of the original hit compounds small molecule inhibitors of Tdp2 with sub-100nM potencies have been identified. This poster will describe our preliminary results in this area.

Abstract 1139: Novel, cellular active inhibitors of G6PD, a key mediator of ROS-induced cellular stress

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway (PPP) and catalyses the oxidation of glucose-6-phosphate to glucono-δ-lactone-6-phosphate with the concomitant production of NADPH. Alongside an important role in the production of biosynthetic precursors such as ribonucleotides, this pathway is a major cellular source of NADPH. As such, the pathway plays a critical role in both the biosynthesis of fatty acids & cholesterol biosynthesis (thus supporting cell division) and maintaining glutathione in its reduced state (GSH), the latter ameliorating cellular stress arising from reactive oxygen species (ROS). Given these key roles, modulation of the pathway has potential therapeutic application, and inhibition of G6PD is an attractive target, particularly in combination with standard of care radiotherapy or cytotoxic drugs which can increase ROS. We have developed a series of novel steroid G6PD inhibitors which, in our hands, modulate flux through the PPP and increase ROS-induced stress in cells. This disclosure will describe some of our results in this area. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1139. doi:1538-7445.AM2012-1139

Abstract 4435: Discovery, biological characterization and oral antitumor activity of polo-like kinase 1 (Plk1) selective small molecule inhibitors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We have identified novel inhibitors of Polo-like kinase 1 (Plk1) that exhibit strong single agent activity in solid tumor and leukemia xenograft models after oral dosing. Plk1 is a serine / threonine protein kinase thought to regulate cell division through promotion of mitotic entry, control of spindle assembly, orchestration of mitotic progression and initiation of cytokinesis. Plk1 has been reported to phosphorylate and deactivate the tumor suppressors p53, p63 and p73 thereby inhibiting apoptosis. Furthermore this repression of p53 family members may be responsible for the survival and tumorigenesis of liver cancer stem cells. Cancer cell proliferation is blocked in vitro and in vivo by antisense oligonucleotides and siRNAs to Plk1. Overexpression of Plk1 is associated with tumor development and many human cancers express elevated Plk1 levels compared to surrounding normal tissue. Numerous studies have shown that Plk1 expression levels correlate with disease progression, invasiveness and poor patient prognosis. Collectively, these observations support the selection of Plk1 as an attractive target for cell cycle-directed cancer therapy. We have employed high throughput screening, in silico screening and de novo ligand design approaches to select an inhibitor scaffold for lead optimization. We have selected a set of ATP-competitive Plk1 inhibitors that exhibit high selectivity for Plk1 and inhibit growth of a broad range of tumor cell lines in vitro with IC50s in the low nanomolar range. Structure-activity relationships (SAR) were determined through iterative targeted analogue synthesis and in vitro testing with SAR rationalized against x-ray crystallographic data. We observed selectivity of the inhibitor scaffold for Plk1 against a panel of over 200 kinases. Treatment of tumor cells with our Plk1 inhibitors induced a phenotype consistent with inhibition of Plk1, accumulation of cells in mitosis and induction of apoptosis. The extent of cytotoxicity was dependent on proliferation as determined by comparative viability of cells arrested in the G1 phase versus proliferating cells. Compound evaluation using a cell-based activity assay for inhibition of Plk1 and pharmacokinetic profiling informed lead optimization studies to generate potential drug candidates with high oral bioavailability and which induce tumor stabilization, regression and tumor-free cures in solid tumor and leukemia xenograft models as single agents. For example, 6% T/C (p = 0.00001) was achieved in the HCT116 human colon carcinoma model. In summary, we describe a set of Plk1 inhibitors which act as potent antiproliferative agents suitable for further development as oral Plk1 selective inhibitors for the treatment of human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4435.