Danny Ganame

Identification of Compounds with Anti-Proliferative Activity against Trypanosoma brucei brucei Strain 427 by a Whole Cell Viability Based HTS Campaign

Human African Trypanosomiasis (HAT) is caused by two trypanosome sub-species, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Drugs available for the treatment of HAT have significant issues related to difficult administration regimes and limited efficacy across species and disease stages. Hence, there is considerable need to find new alternative and less toxic drugs. An approach to identify starting points for new drug candidates is high throughput screening (HTS) of large compound library collections. We describe the application of an Alamar Blue based, 384-well HTS assay to screen a library of 87,296 compounds against the related trypanosome subspecies, Trypanosoma brucei brucei bloodstream form lister 427. Primary hits identified against T.b. brucei were retested and the IC50 value compounds were estimated for T.b. brucei and a mammalian cell line HEK293, to determine a selectivity index for each compound. The screening campaign identified 205 compounds with greater than 10 times selectivity against T.b. brucei. Cluster analysis of these compounds, taking into account chemical and structural properties required for drug-like compounds, afforded a panel of eight compounds for further biological analysis. These compounds had IC50 values ranging from 0.22 µM to 4 µM with associated selectivity indices ranging from 19 to greater than 345. Further testing against T.b. rhodesiense led to the selection of 6 compounds from 5 new chemical classes with activity against the causative species of HAT, which can be considered potential candidates for HAT early drug discovery. Structure activity relationship (SAR) mining revealed components of those hit compound structures that may be important for biological activity. Four of these compounds have undergone further testing to 1) determine whether they are cidal or static in vitro at the minimum inhibitory concentration (MIC), and 2) estimate the time to kill.

Total synthesis of 8-deshydroxyajudazol B.

The total synthesis of a stereoisomer of 8-deshydroxyajudazol B (4), the putative biosynthetic intermediate of the ajudazols A (1) and B (2), is described. The key steps in the synthesis included an intramolecular Diels-Alder (IMDA) reaction to secure the isochromanone fragment, a novel selective acylation/O,N-shift to give a hydroxyamide which was cyclized to the oxazole and a high yielding Sonogashira coupling to form the C18-C19 bond. Partial alkyne reduction then afforded the target 4.

Total synthesis of the proposed structure of 8-deshydroxyajudazol A: a modified approach to 2,4-disubstituted oxazoles.

The total synthesis of the proposed structure for the minor myxobacterial metabolite 8-deshydroxyajudazol A (3) is described. The isochromanone moiety present in the eastern fragment was constructed by an intramolecular-Diels-Alder (IMDA). Difficulties were encountered with the formation of the 2,4-disubstituted oxazole, so this was synthesized via a modified approach. This involved selective acylation of the diol 7 with acid 8, azide displacement of the secondary alcohol, and subsequent azide reduction in the presence of base which induced an O,N shift to give the hydroxyamide 23. Cyclodehydration then gave the desired oxazole 24 and deprotection followed by mesylation and elimination produced the C15 alkene 5. Sonogashira coupling with the eastern fragment vinyl iodide 6 and partial reduction yielded 8-deshydroxyajudazol A (3).

6-Arylpyrazine-2-carboxamides : a new core for Trypanosoma brucei inhibitors

From a whole-organism high throughput screen of approximately 87000 compounds against Trypanosoma brucei brucei, we recently identified eight new unique compounds for the treatment of human African trypanosomiasis. In an effort to understand the structure-activity relationships around these compounds, we report for the first time our results on a new class of trypanocides, the pyrazine carboxamides. Attracted by the low molecular weight (270 g·mol(-1)) of our starting hit (9) and its potency (0.49 μM), the SAR around the core was explored, leading to compounds having an EC50 as low as 25 nM against T. b. brucei and being more than 1500 times less toxic against mammalian L6 and HEK293 cell lines. The most potent compounds in the series were exquisitely selective for T. brucei over a panel of other protozoan parasites, showing an excellent correlation with the human infective parasite Trypanosoma brucei rhodesiense, the most potent compound (65) having an EC50 of 24 nM. The compounds are highly drug-like and are able to penetrate the CNS, their only limitation currently being their rate of microsomal metabolism. To that effect, efforts to identify potential metabolites of selected compounds are also reported.

Development of substituted 7-phenyl-4-aminobenzothieno[3,2-d] pyrimidines as potent LIMK1 inhibitors

7-Phenyl-4-aminobenzothieno[3,2-d]pyrimidines were previously reported to exhibit moderate LIMK1 inhibition. Further exploration of SAR around the 7-phenyl moiety has led to the development of a lead series with an increased potency for LIMK1. Evaluation of physicochemical and ADME properties, and off-target kinase screens has seen this novel series emerge as a promising platform for a set of tool compounds for evaluating LIMK as a therapeutic target.

Abstract 5371: PRMT5 inhibitors as novel treatment for cancers

Increased expression or dysregulation of protein arginine methyltransferase 5 (PRMT5) activity is associated with poor prognosis in many cancers. Through increased methylation of epigenetic and non-epigenetic targets, the aberrant activity of PRMT5 has been associated with many pro-tumourigenic cellular changes such as, increased levels of protein synthesis, dysregulation of cell cycle, cellular adaptation to hypoxic conditions, and suppression of normal cell death pathways. Genetic studies suggest that suppression of PRMT5 activity can reverse many of these pro-tumourigenic effects making PRMT5 an attractive drug discovery target. We screened a library of 350,000 lead-like compounds with a biochemical assay measuring the methylation of a histone H4 peptide by the recombinant human PRMT5/MEP50 complex. Biochemical and biophysical profiling of the inhibitory compounds indicated that several distinct binding modes were exhibited by the different chemical scaffolds. Inhibitors displayed competitive, noncompetitive or uncompetitive interactions with respect to S-adenosyl methionine and the peptide substrate. Medicinal chemistry developed several classes of potent, highly selective inhibitors of PRMT5 methyltransferase activity from the hit set. The optimised tool compound, CTx-034, is a potent inhibitor of PRMT5 methyl transferase activity (KD = 2 nM), which is highly selective (>100-fold) versus a panel of 18 methyltransferases (including 6 PRMT family members), 11 lysine demethylases, and 15 safety related targets (GPCRs, ion channels, enzymes). Treatment of cancer cell lines with CTx-034 reduces cellular levels of symmetrically dimethylated H4 Arginine 3 (H4R3me2s), in a dose dependent manner (IC50 = 4 nM) to levels undetectable by Western blot. Furthermore, within this chemical series the ability of compounds to reduce cellular levels of H4R3me2s closely correlates with PRMT5 inhibitory activity supporting PRMT5 as the cellular target of these compounds, and suggesting that PRMT5 is the major writer of this histone mark in many cancer cell lines. CTx-034 also inhibits the symmetric dimethylation of arginine on other histone and non-histone cellular substrates of PRMT5, including H3R2me2s and SmD1. Conversely, CTx-034 treatment does not reduce levels of H4R3 asymmetric dimethylation, a histone mark catalysed by PRMT1. Finally, CTx-034 has good oral bioavailability and pharmacokinetic properties in rodents and twice-daily dosing (10 - 100 mg/kg) over 10-14 days produces a dose dependent reduction of the H4R3me2s mark in bone marrow cells and peripheral white blood cells. This treatment is well tolerated by the mice, with no significant reduction in body weight or changes in haematological parameters observed. CTx-034 provides an excellent tool compound for cellular and in vivo proof of concept studies. Citation Format: Hendrik Falk, Richard C. Foitzik, Elizabeth Allan, Melanie deSilva, Hong Yang, Ylva E. Bozikis, Marica Nikac, Scott R. Walker, Michelle A. Camerino, Ben J. Morrow, Alexandra E. Stupple, Rachel Lagiakos, Jo-Anne Pinson, Romina Lessene, Wilhelmus JA Kersten, Danny G. Ganame, Ian P. Holmes, Gill E. Lunniss, Matthew Chung, Stefan J. Hermans, Michael W. Parker, Alison Thistlethwaite, Karen White, Susan A. Charman, Brendon J. Monahan, Patricia Pilling, Julian Grusovin, Thomas S. Peat, Stefan Sonderegger, Emma Toulmin, Stephen M. Jane, David J. Curtis, Paul A. Stupple, Ian P. Street. PRMT5 inhibitors as novel treatment for cancers. [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 5371. doi:10.1158/1538-7445.AM2015-5371

Abstract 4029: BL-011256 is a novel VEGFR3 selective inhibitor, which suppresses tumor lymphatics and lymph node metastasis in an animal model of melanoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The role of VEGFR3 in lymphangiogenesis has been well established. Targeting VEGFR3 has been shown to curtail tumor progression mediated via lymphatic dissemination. More recently VEGFR3 was shown to play an important role in the mediation of tumor-induced immune cell tolerance. We have identified BL-011256, a novel inhibitor of VEGFR3 that exhibits 17-fold selectivity over VEGFR2 and a narrow tyrosine kinase inhibition spectrum. A 7-day b.i.d repeat oral dose study showed that BL-011256 is well tolerated in mice. Mouse plasma exposure experiments demonstrated that BL-011256 attains free drug plasma concentration levels that exceed the concentration required for IC50 activity on VEGFR3 in vitro but are considerably lower than the in vitro IC50 required for activity on VEGFR2. In the B16F10 mouse melanoma model, animals bearing melanoma tumors displayed considerably attenuated signs of tumor progression when treated with BL-011256. BL-011256 caused a 70% reduction in primary lesion growth and a 50% reduction in metastasis to the draining lymph node. Furthermore, BL-011256 was active in reducing the number of satellite in-transit metastases. Immunohistochemical whole mount analyses on ears with primary tumor lesions derived from BL-011256 treated and vehicle-treated mice using Lyve-1 for the identification of lymphatic vessels and CD31 for the identification of blood vessels was conducted. Tumors in vehicle-treated mice displayed a peri-tumoral area densely populated by lymphatic vessels. In contrast, tumors derived from BL-011256 treated mice were devoid of peri-tumoral lymphatic vessels. Notably, both vehicle-treated and BL-011256-treated animals displayed similar staining for peritumoral blood vessels, suggesting no effect on blood vessels (this is consistent with no activity on VEGFR2). Furthermore, PK sampling during the last day of dosing in a 14-day dosing schedule demonstrated that there is no compound accumulation during the repeat dosing schedule utilised in the B16F10 tumour efficacy experiment. In conclusion BL-011256 has been identified as a selective inhibitor of VEGFR3 that supresses both primary tumor growth and lymph node metastasis. Citation Format: Annabell Leske, Richard Foitzik, Donna Beaumont, John Bentley, Ylva Bergman, Chloe Brown, Michelle Camerino, Susan Charman, Neil Choi, Melanie De Silva, Matthew Chung, Hendrik Falk, Danny Ganame, Alison Gregg, Julian Grusovin, Andrew Harvey, Catherine Hemley, Ian Holmes, Belinda Huff, Daniel Inglis, Wilhelmus Kersten, Tina Lavranos, Romina Lessene, Gillian Lunniss, Brendon Monahan, Benjamin Morrow, Marica Nikac, George Nikolakopoulos, Dharam Paul, Tom Peat, Justin Ripper, Michaela Scherer, Paul Stupple, Karen White, Ian Street, Gabriel Kremmidiotis. BL-011256 is a novel VEGFR3 selective inhibitor, which suppresses tumor lymphatics and lymph node metastasis in an animal model of melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4029. doi:10.1158/1538-7445.AM2014-4029