Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase.

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.

3q26-29 Amplification in head and neck squamous cell carcinoma: a review of established and prospective oncogenes.

Head and neck squamous cell carcinoma (HNSCC) is significantly underrepresented in worldwide cancer research, yet survival rates for the disease have remained static for over 50 years. Distant metastasis is often present at the time of diagnosis, and is the primary cause of death in cancer patients. In the absence of routine effective targeted therapies, the standard of care treatment remains chemoradiation in combination with (often disfiguring) surgery. A defining characteristic of HNSCC is the amplification of a region of chromosome 3 (3q26-29), which is consistently associated with poorer patient outcome. This review provides an overview of the role the 3q26-29 region plays in HNSCC, in terms of both known and as yet undiscovered processes, which may have potential clinical relevance.

The High-Affinity Interaction between ORC and DNA that Is Required for Replication Licensing Is Inhibited by 2-Arylquinolin-4-Amines.

In late mitosis and G1, origins of DNA replication must be "licensed" for use in the upcoming S phase by being encircled by double hexamers of the minichromosome maintenance proteins MCM2-7. A "licensing checkpoint" delays cells in G1 until sufficient origins have been licensed, but this checkpoint is lost in cancer cells. Inhibition of licensing can therefore kill cancer cells while only delaying normal cells in G1. In a high-throughput cell-based screen for licensing inhibitors we identified a family of 2-arylquinolin-4-amines, the most potent of which we call RL5a. The binding of the origin recognition complex (ORC) to origin DNA is the first step of the licensing reaction. We show that RL5a prevents ORC forming a tight complex with DNA that is required for MCM2-7 loading. Formation of this ORC-DNA complex requires ATP, and we show that RL5a inhibits ORC allosterically to mimic a lack of ATP.

Strategic siRNA screening approaches to target cancer at the Cancer Research UK Beatson Institute.

The RNAi Screening Facility at the Cancer Research UK Beatson Institute combines siRNA genome-wide screening with drug screening coupled with High Content Imaging and fluorescence-based phenotypic assays to target multiple types of cancer. Here, we describe the infrastructure of the Facility and the approaches we utilise. We also share our experiences in running such a facility and developing and executing screening campaigns, with particular regard to high content multiparametric analysis, data management and statistical analysis.

Dihydroquinazolines as a novel class of Trypanosoma brucei trypanothione reductase inhibitors: discovery, synthesis, and characterization of their binding mode by protein crystallography.

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.

Design, synthesis and biological evaluation of novel inhibitors of Trypanosoma brucei pteridine reductase 1.

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 (K(i)(app)=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 K(m) to K(i) ratio (K(m)=25 nM and K(i)=2.3 nM, respectively).

Synthesis and evaluation of 1-(1-(Benzo[b]thiophen-2-yl)cyclohexyl)piperidine (BTCP) analogues as inhibitors of trypanothione reductase.

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 (K(i)=1 microM) and biologically active against bloodstream T. brucei (EC(50)=10 microM), but with poor selectivity against mammalian MRC5 cells (EC(50)=29 microM). Analogues with improved enzymatic and biological activity were obtained. The structure-activity relationships of this novel series are discussed.

Investigation of trypanothione reductase as a drug target in Trypanosoma brucei.

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 62,000 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.

One scaffold, three binding modes: novel and selective pteridine reductase 1 inhibitors derived from fragment hits discovered by virtual screening.

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.