David G. Lloyd

Comprehensive characterization of cytochrome P450 isozyme selectivity across chemical libraries

The cytochrome P450 (CYP) gene family catalyzes drug metabolism and bioactivation and is therefore relevant to drug development. We determined potency values for 17,143 compounds against five recombinant CYP isozymes (1A2, 2C9, 2C19, 2D6 and 3A4) using an in vitro bioluminescent assay. The compounds included libraries of US Food and Drug Administration (FDA)-approved drugs and screening libraries. We observed cross-library isozyme inhibition (30–78%) with important differences between libraries. Whereas only 7% of the typical screening library was inactive against all five isozymes, 33% of FDA-approved drugs were inactive, reflecting the optimized pharmacological properties of the latter. Our results suggest that low CYP 2C isozyme activity is a common property of drugs, whereas other isozymes, such as CYP 2D6, show little discrimination between drugs and unoptimized compounds found in screening libraries. We also identified chemical substructures that differentiated between the five isozymes. The pharmacological compendium described here should further the understanding of CYP isozymes.

Synthesis and Evaluation of Azetidinone Analogues of Combretastatin A-4 as Tubulin Targeting Agents

The synthesis and antiproliferative activity of a new series of rigid analogues of combretastatin A-4 are described which contain the 1,4-diaryl-2-azetidinone (β-lactam) ring system in place of the usual ethylene bridge present in the natural combretastatin stilbene products. These novel compounds are also substituted at position 3 of the β-lactam ring with an aryl ring. A number of analogues showed potent nanomolar activity in human MCF-7 and MDA-MB-231 breast cancer cell lines, displayed in vitro inhibition of tubulin polymerization, and did not cause significant cytotoxicity in normal murine breast epithelial cells. 4-(4-Methoxyaryl)-substituted compound 32, 4-(3-hydroxy-4-methoxyaryl)-substituted compounds 35 and 41, and the 3-(4-aminoaryl)-substituted compounds 46 and 47 displayed the most potent antiproliferative activity of the series. β-Lactam 41 in particular showed subnanomolar activity in MCF-7 breast cancer cells (IC₅₀= 0.8 nM) together with significant in vitro inhibition of tubulin polymerization and has been selected for further biochemical assessment. These novel β-lactam compounds are identified as potentially useful scaffolds for the further development of antitumor agents that target tubulin.

Screening for Inhibitors of Tau Protein Aggregation into Alzheimer Paired Helical Filaments: A Ligand Based Approach Results in Successful Scaffold Hopping

The aggregation of tau protein into paired helical filaments is one of the hallmarks of Alzheimers disease and related dementias. We therefore continue our search for non-toxic, cell penetrating inhibitors of tau aggregation, which hold potential for brain penetration. Pickhardt et al. (2005) have reported a high throughput screen for tau aggregation inhibitors previously, which resulted in the identification of several hit classes. Here we report the identification of novel inhibitors which were not present in the initial high throughput assay. This was achieved by transformation of the high throughput screen data into the 3D relationships of virtual pharmacophores The pharmacophore models were utilized in a virtual screen of a Maybridge database. The virtual screen provided 136 hits; 19 representative hits were selected and assayed, this resulted in two novel leads with an IC(50) < 13 microM. These two leads feature a novel scaffold for tau aggregation inhibitors.

Lead identification of conformationally restricted β-lactam type combretastatin analogues: Synthesis, antiproliferative activity and tubulin targeting effects

The synthesis and study of the structure-activity relationships of a series of rigid analogues of combretastatin A-4 are described which contain the 1,4-diaryl-2-azetidinone (β-lactam) ring system in place of the usual ethylene bridge present in the natural combretastatin stilbene products. The 1,4-diaryl-2-azetidinones are unsubstituted at C-3, or contain methyl substituent(s) at C-3. The most potent compounds 12d and 12e display antiproliferative activity at nanomolar concentrations when evaluated against the MCF-7 and MDA-MB-231 human breast carcinoma cell lines. 12d exerts antimitotic effects through an inhibition of tubulin polymerisation and subsequent G2/M arrest of the cell cycle in human MDA-MB-231 breast cancer cells, with similar activity to that of CA-4. These novel β-lactam compounds are identified as potentially useful scaffolds for the further development of antitumour agents which target tubulin.

Synthesis, Evaluation and Structural Studies of Antiproliferative Tubulin-targeting Azetidin-2-ones

A series of azetidin-2-ones substituted at positions 1, 3 and 4 of the azetidinone ring scaffold were synthesised and evaluated for antiproliferative, cytotoxic and tubulin-binding activity. In these compounds, the cis double bond of the vascular targeting agent combretastatin A-4 is replaced with the azetidinone ring in order to enhance the antiproliferative effects displayed by combretastatin A-4 and prevent the cis/trans isomerisation that is associated with inactivation of combretastatin A-4. The series of azetidinones was synthetically accessible via the Staudinger and Reformatsky reactions. Of a diverse range of heterocyclic derivatives, 3-(2-thienyl) analogue 28 and 3-(3-thienyl) analogue 29 displayed the highest potency in human MCF-7 breast cancer cells with IC(50) values of 7 nM and 10nM, respectively, comparable to combretastatin A-4. Compounds from this series also exhibited potent activity in MDA-MB-231 breast cancer cells and in the NCI60 cell line panel. No significant toxicity was observed in normal murine breast epithelial cells. The presence of larger, bulkier groups at the 3-position, for example, 3-naphthyl derivative 21 and 3-benzothienyl derivative 26, resulted in relatively lower antiproliferative activity in the micromolar range. Tubulin-binding studies of 28 (IC(50)=1.37 μM) confirmed that the molecular target of this series of compounds is tubulin. These novel 3-(thienyl) β-lactam antiproliferative agents are useful scaffolds for the development of tubulin-targeting drugs.

Beyond the Ligand‐Binding Pocket: Targeting Alternate Sites in Nuclear Receptors

Nuclear receptors (NRs) are a family of ligand‐modulated transcription factors with significant therapeutic relevance from metabolic disorders and inflammation to cancer, neurodegenerative, and psychiatric disorders. Drug discovery efforts are typically concentrated on modulating the natural ligand action within the ligand‐binding pocket (LBP) in the C‐terminal ligand‐binding domain (LBD). Drawbacks of LBP‐based strategies include physiological alterations due to disruption of ligand binding and difficulties in achieving tissue specificity. Furthermore, the lack of a “pure” and predictable mechanism of action predisposes such intervention toward drug resistance. Recent outstanding progress in our understanding of NR biology has shifted the focus of drug discovery efforts from inside to outside the LBP, affording consideration to the interaction between NRs and coactivator proteins, the interaction between NRs and DNA and the NRs’ ligand‐independent functions. This review encompasses such currently available NR non‐LBP‐based interventions and their potential application in therapy or as specific tools to probe NR biology.

The effect of tightly bound water molecules on the structural interpretation of ligand-derived pharmacophore models

The importance of the consideration of water molecules in the structural interpretation of ligand-derived pharmacophore models is explored. We compare and combine results from recently introduced methods for bound-water molecule identification in protein binding sites and ligand-superposition-based pharmacophore derivation, for the interpretation of ligand-derived pharmacophore models. In the analysis of thymidine kinase (HSV-1) and poly (ADP-ribose) polymerase (PARP), the concurrent application of both methods leads to an agreement in the prediction of tightly bound water molecules as key pharmacophoric points in the binding site of these proteins. This agreement has implications for approaching binding site analysis and consensus drug design, as it highlights how pharmacophore-based models of binding sites can include interaction features not only with protein groups but also with bound water molecules.

True antiandrogens-selective non-ligand-binding pocket disruptors of androgen receptor-coactivator interactions: novel tools for prostate cancer.

Prostate cancer (PCa) therapy typically involves administration of “classical” antiandrogens, competitive inhibitors of androgen receptor (AR) ligands, dihydrotestosterone (DHT) and testosterone (tes), for the ligand-binding pocket (LBP) in the ligand-binding domain (LBD) of AR. Prolonged LBP-targeting leads to resistance, and alternative therapies are urgently required. We report the identification and characterization of a novel series of diarylhydrazides as selective disruptors of AR interaction with coactivators through application of structure and ligand-based virtual screening. Compounds demonstrate full (“true”) antagonism in AR with low micromolar potency, selectivity over estrogen receptors α and β and glucocorticoid receptor, and partial antagonism of the progesterone receptor. MDG506 (5) demonstrates low cellular toxicity in PCa models and dose responsive reduction of classical antiandrogen-induced prostate specific antigen expression. These data provide compelling evidence for such non-LBP intervention as an alternative approach or in combination with classical PCa therapy.

Synthesis, biological evaluation, structural-activity relationship, and docking study for a series of benzoxepin-derived estrogen receptor modulators.

The estrogen receptors ERalpha and ERbeta are recognized as important pharmaceutical targets for a variety of diseases including osteoporosis and breast cancer. A series of novel benzoxepin-derived compounds are described as potent selective modulators of the human estrogen receptor modulators (SERMs). We report the antiproliferative effects of these compounds on human MCF-7 breast tumor cells. These heterocyclic compounds contain the triarylethylene arrangement as exemplified by tamoxifen, conformationally restrained through the incorporation of the benzoxepin ring system. The compounds demonstrate potency at nanomolar concentrations in antiproliferative assays against an MCF-7 human breast cancer cell line with low cytotoxicity together with low nanomolar binding affinity for the estrogen receptor. The compounds also demonstrate potent antiestrogenic properties in the human uterine Ishikawa cell line. The effect of a number of functional group substitutions on the ER binding properties of the benzoxepin molecular scaffold is examined through a detailed docking and 2D-QSAR computational investigation. The best QSAR model developed for ERalphabeta selectivity yielded R(2) of 0.84 with an RMSE for the training set of 0.30. The predictive quality of the model was Q(2) of 0.72 and RMSE of 0.18 for the test set. One particular compound bearing a 4-fluoro substituent, exhibits 15-fold selectivity for ERbeta and both our docking and QSAR studies converge on the correlation between enhanced lipophilicity and enhanced ERbeta binding for this benzoxepin ring scaffold.

Synthesis, biochemical and molecular modelling studies of antiproliferative azetidinones causing microtubule disruption and mitotic catastrophe

The structure-activity relationships of antiproliferative β-lactams, focusing on modifications at the 4-position of the β-lactam ring, is described. Synthesis of this series of compounds was achieved utilizing the Staudinger and Reformatsky reactions. The antiproliferative activity was assessed in MCF-7 cells, where the 4-(4-ethoxy)phenyl substituted compound 26 displayed the most potent activity with an IC(50) value of 0.22 μM. The mechanism of action was demonstrated to be by inhibition of tubulin polymerisation. Cell exposure to combretastatin A-4 and 26 led to arrest of MCF-7 cells in the G2/M phase of the cell cycle and induction of apoptosis. Additionally, mitotic catastrophe for combretastatin A-4 and for 26 was demonstrated in breast cancer cells for the first time, as evidenced by the formation of giant, multinucleated cells.