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1.  Acetyl-CoA Synthetase 2 Promotes Acetate Utilization and Maintains Cancer Cell Growth under Metabolic Stress 
Cancer Cell  2015;27(1):57-71.
A functional genomics study revealed that the activity of acetyl-CoA synthetase 2 (ACSS2) contributes to cancer cell growth under low-oxygen and lipid-depleted conditions. Comparative metabolomics and lipidomics demonstrated that acetate is used as a nutritional source by cancer cells in an ACSS2-dependent manner, and supplied a significant fraction of the carbon within the fatty acid and phospholipid pools. ACSS2 expression is upregulated under metabolically stressed conditions and ACSS2 silencing reduced the growth of tumor xenografts. ACSS2 exhibits copy-number gain in human breast tumors, and ACSS2 expression correlates with disease progression. These results signify a critical role for acetate consumption in the production of lipid biomass within the harsh tumor microenvironment.
Graphical Abstract
•ACSS2 expression positively correlates with tumor stage and patient survival•Hypoxia and low lipid availability synergistically stimulate ACSS2 expression•Acetate is a major source of carbon for lipid synthesis during metabolic stress•ACSS2 is required for growth of tumor xenografts harboring ACSS2 copy-number gains
Schug et al. show that ACSS2 expression is increased in cancer cells under metabolic stress, and it is critical for cancer cells to use acetate as a nutritional source for lipid biomass production under this condition. Importantly, the ACSS2 expression level correlates with breast cancer progression.
PMCID: PMC4297291  PMID: 25584894
2.  Statistical Methods for Analysis of High-Throughput RNA Interference Screens 
Nature methods  2009;6(8):569-575.
RNA interference (RNAi) has become a powerful technique for reverse genetics and drug discovery and, in both of these areas, large-scale high-throughput RNAi screens are commonly performed. The statistical techniques used to analyze these screens are frequently borrowed directly from small-molecule screening; however small-molecule and RNAi data characteristics differ in meaningful ways. We examine the similarities and differences between RNAi and small-molecule screens, highlighting particular characteristics of RNAi screen data that must be addressed during analysis. Additionally, we provide guidance on selection of analysis techniques in the context of a sample workflow.
PMCID: PMC2789971  PMID: 19644458
3.  Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase 
Analytical Biochemistry  2010;396(2):194-203.
Activity of the pterin- and folate-salvaging enzymes pteridine reductase 1 (PTR1) and dihydrofolate reductase–thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS.
PMCID: PMC2789237  PMID: 19748480
Drug discovery; Screening; Pteridine reductase; Dihydrofolate reductase
4.  Investigation of Trypanothione Reductase as a Drug Target in Trypanosoma brucei 
Chemmedchem  2009;4(12):2060-2069.
There is an urgent need for new drugs for the treatment of tropical parasitic diseases such as human African trypanosomiasis, which is caused by Trypanosoma brucei. The enzyme trypanothione reductase (TryR) is a potential drug target within these organisms. Herein we report the screening of a 62000 compound library against T. brucei TryR. Further work was undertaken to optimise potency and selectivity of two novel-compound series arising from the enzymatic and whole parasite screens and mammalian cell counterscreens. Both of these series, containing either a quinoline or pyrimidinopyrazine scaffold, yielded low micromolar inhibitors of the enzyme and growth of the parasite. The challenges of inhibiting TryR with druglike molecules is discussed.
PMCID: PMC2855869  PMID: 19924760
human African trypanosomiasis; pyrimidopyridazines; quinolines; trypanosoma brucei; trypanothione reductase
5.  Synthesis and Evaluation of 1-(1-(Benzo[b]thiophen-2-yl)cyclohexyl)piperidine (BTCP) Analogues as Inhibitors of Trypanothione Reductase 
Chemmedchem  2009;4(8):1341-1353.
Thirty two analogues of phencyclidine were synthesised and tested as inhibitors of trypanothione reductase (TryR), a potential drug target in trypanosome and leishmania parasites. The lead compound BTCP (1, 1-(1-benzo[b]thiophen-2-yl-cyclohexyl) piperidine) was found to be a competitive inhibitor of the enzyme (Ki=1 μm) and biologically active against bloodstream T. brucei (EC50=10 μm), but with poor selectivity against mammalian MRC5 cells (EC50=29 μm). Analogues with improved enzymatic and biological activity were obtained. The structure–activity relationships of this novel series are discussed.
PMCID: PMC2929374  PMID: 19557802
BTCP; inhibitors; medicinal chemistry; trypanosoma brucei; trypanothione reductase
6.  One Scaffold, Three Binding Modes: Novel and Selective Pteridine Reductase 1 Inhibitors Derived from Fragment Hits Discovered by Virtual Screening† 
Journal of Medicinal Chemistry  2009;52(14):4454-4465.
The enzyme pteridine reductase 1 (PTR1) is a potential target for new compounds to treat human African trypanosomiasis. A virtual screening campaign for fragments inhibiting PTR1 was carried out. Two novel chemical series were identified containing aminobenzothiazole and aminobenzimidazole scaffolds, respectively. One of the hits (2-amino-6-chloro-benzimidazole) was subjected to crystal structure analysis and a high resolution crystal structure in complex with PTR1 was obtained, confirming the predicted binding mode. However, the crystal structures of two analogues (2-amino-benzimidazole and 1-(3,4-dichloro-benzyl)-2-amino-benzimidazole) in complex with PTR1 revealed two alternative binding modes. In these complexes, previously unobserved protein movements and water-mediated protein−ligand contacts occurred, which prohibited a correct prediction of the binding modes. On the basis of the alternative binding mode of 1-(3,4-dichloro-benzyl)-2-amino-benzimidazole, derivatives were designed and selective PTR1 inhibitors with low nanomolar potency and favorable physicochemical properties were obtained.
PMCID: PMC2966039  PMID: 19527033
7.  Design, Synthesis and Biological Evaluation of Novel Inhibitors of Trypanosoma brucei Pteridine Reductase 1 
Chemmedchem  2010;6(2):302-308.
Genetic studies indicate that the enzyme pteridine reductase 1 (PTR1) is essential for the survival of the protozoan parasite Trypanosoma brucei. Herein, we describe the development and optimisation of a novel series of PTR1 inhibitors, based on benzo[d]imidazol-2-amine derivatives. Data are reported on 33 compounds. This series was initially discovered by a virtual screening campaign (J. Med. Chem., 2009, 52, 4454). The inhibitors adopted an alternative binding mode to those of the natural ligands, biopterin and dihydrobiopterin, and classical inhibitors, such as methotrexate. Using both rational medicinal chemistry and structure-based approaches, we were able to derive compounds with potent activity against T. brucei PTR1 (=7 nm), which had high selectivity over both human and T. brucei dihydrofolate reductase. Unfortunately, these compounds displayed weak activity against the parasites. Kinetic studies and analysis indicate that the main reason for the lack of cell potency is due to the compounds having insufficient potency against the enzyme, which can be seen from the low Km to Ki ratio (Km=25 nm and Ki=2.3 nm, respectively).
PMCID: PMC3047710  PMID: 21275054
antiprotozoal agents; drug discovery; pteridine reductase; structure-based drug design; Trypanosoma brucei
8.  Dihydroquinazolines as a Novel Class of Trypanosoma brucei Trypanothione Reductase Inhibitors: Discovery, Synthesis, and Characterization of their Binding Mode by Protein Crystallography 
Journal of Medicinal Chemistry  2011;54(19):6514-6530.
Trypanothione reductase (TryR) is a genetically validated drug target in the parasite Trypanosoma brucei, the causative agent of human African trypanosomiasis. Here we report the discovery, synthesis, and development of a novel series of TryR inhibitors based on a 3,4-dihydroquinazoline scaffold. In addition, a high resolution crystal structure of TryR, alone and in complex with substrates and inhibitors from this series, is presented. This represents the first report of a high resolution complex between a noncovalent ligand and this enzyme. Structural studies revealed that upon ligand binding the enzyme undergoes a conformational change to create a new subpocket which is occupied by an aryl group on the ligand. Therefore, the inhibitor, in effect, creates its own small binding pocket within the otherwise large, solvent exposed active site. The TryR–ligand structure was subsequently used to guide the synthesis of inhibitors, including analogues that challenged the induced subpocket. This resulted in the development of inhibitors with improved potency against both TryR and T. brucei parasites in a whole cell assay.
PMCID: PMC3188286  PMID: 21851087

Results 1-8 (8)