Roderick E. Hubbard

Molecular basis of agonism and antagonism in the oestrogen receptor.

Oestrogens are involved in the growth, development and homeostasis of a number of tissues. The physiological effects of these steroids are mediated by a ligand-inducible nuclear transcription factor, the oestrogen receptor (ER). Hormone binding to the ligand-binding domain (LBD) of the ER initiates a series of molecular events culminating in the activation or repression of target genes. Transcriptional regulation arises from the direct interaction of the ER with components of the cellular transcription machinery,. Here we report the crystal structures of the LBD of ER in complex with the endogenous oestrogen, 17β-oestradiol, and the selective antagonist raloxifene, at resolutions of 3.1 and 2.6 Å, respectively. The structures provide a molecular basis for the distinctive pharmacophore of the ER and its catholic binding properties. Agonist and antagonist bind at the same site within the core of the LBD but demonstrate different binding modes. In addition, each class of ligand induces a distinct conformation in the transactivation domain of the LBD, providing structural evidence of the mechanism of antagonism.

Structure of the Ligand-Binding Domain of Oestrogen Receptor Beta in the Presence of a Partial Agonist and a Full Antagonist

Oestrogens exert their physiological effects through two receptor subtypes. Here we report the three‐dimensional structure of the oestrogen receptor beta isoform (ERβ) ligand‐binding domain (LBD) in the presence of the phyto‐oestrogen genistein and the antagonist raloxifene. The overall structure of ERβ‐LBD is very similar to that previously reported for ERα. Each ligand interacts with a unique set of residues within the hormone‐binding cavity and induces a distinct orientation in the AF‐2 helix (H12). The bulky side chain of raloxifene protrudes from the cavity and physically prevents the alignment of H12 over the bound ligand. In contrast, genistein is completely buried within the hydrophobic core of the protein and binds in a manner similar to that observed for ERs endogenous hormone, 17β‐oestradiol. However, in the ERβ–genistein complex, H12 does not adopt the distinctive ’agonist‘ position but, instead, lies in a similar orientation to that induced by ER antagonists. Such a sub‐optimal alignment of the transactivation helix is consistent with genisteins partial agonist character in ERβ and demonstrates how ERs transcriptional response to certain bound ligands is attenuated.

NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

We describe the biological properties of NVP-AUY922, a novel resorcinylic isoxazole amide heat shock protein 90 (HSP90) inhibitor. NVP-AUY922 potently inhibits HSP90 (K(d) = 1.7 nmol/L) and proliferation of human tumor cells with GI(50) values of approximately 2 to 40 nmol/L, inducing G(1)-G(2) arrest and apoptosis. Activity is independent of NQO1/DT-diaphorase, maintained in drug-resistant cells and under hypoxic conditions. The molecular signature of HSP90 inhibition, comprising induced HSP72 and depleted client proteins, was readily demonstrable. NVP-AUY922 was glucuronidated less than previously described isoxazoles, yielding higher drug levels in human cancer cells and xenografts. Daily dosing of NVP-AUY922 (50 mg/kg i.p. or i.v.) to athymic mice generated peak tumor levels at least 100-fold above cellular GI(50). This produced statistically significant growth inhibition and/or regressions in human tumor xenografts with diverse oncogenic profiles: BT474 breast tumor treated/control, 21%; A2780 ovarian, 11%; U87MG glioblastoma, 7%; PC3 prostate, 37%; and WM266.4 melanoma, 31%. Therapeutic effects were concordant with changes in pharmacodynamic markers, including induction of HSP72 and depletion of ERBB2, CRAF, cyclin-dependent kinase 4, phospho-AKT/total AKT, and hypoxia-inducible factor-1alpha, determined by Western blot, electrochemiluminescent immunoassay, or immunohistochemistry. NVP-AUY922 also significantly inhibited tumor cell chemotaxis/invasion in vitro, WM266.4 melanoma lung metastases, and lymphatic metastases from orthotopically implanted PC3LN3 prostate carcinoma. NVP-AUY922 inhibited proliferation, chemomigration, and tubular differentiation of human endothelial cells and antiangiogenic activity was reflected in reduced microvessel density in tumor xenografts. Collectively, the data show that NVP-AUY922 is a potent, novel inhibitor of HSP90, acting via several processes (cytostasis, apoptosis, invasion, and angiogenesis) to inhibit tumor growth and metastasis. NVP-AUY922 has entered phase I clinical trials.

Structural Insights into the Mode of Action of a Pure Antiestrogen

BACKGROUND Estrogens exert their effects on target tissues by binding to a nuclear transcription factor termed the estrogen receptor (ER). Previous structural studies have demonstrated that each class of ER ligand (agonist, partial agonist, and SERM antagonist) induces distinctive orientations in the receptors carboxy-terminal transactivation helix. The conformation of this portion of the receptor determines whether ER can recruit and interact with the components of the transcriptional machinery, thereby facilitating target gene expression. RESULTS We have determined the structure of rat ERbeta ligand binding domain (LBD) in complex with the pure antiestrogen ICI 164,384 at 2.3 A resolution. The binding of this compound to the receptor completely abolishes the association between the transactivation helix (H12) and the rest of the LBD. The structure reveals that the terminal portion of ICIs bulky side chain substituent protrudes from the hormone binding pocket, binds along the coactivator recruitment site, and physically prevents H12 from adopting either its characteristic agonist or AF2 antagonist orientation. CONCLUSIONS The binding mode adopted by the pure antiestrogen is similar to that seen for other ER antagonists. However, the size and resultant positioning of the ligands side chain substituent produces a receptor conformation that is distinct from that adopted in the presence of other classes of ER ligands. The novel observation that binding of ICI results in the complete destabilization of H12 provides some indications as to a possible mechanism for pure receptor antagonism.

A structural biologist's view of the oestrogen receptor.

Here we review the results that have emerged from our structural studies on the oestrogen receptor ligand-binding domain (ER-LBD). The effects of agonists and antagonists on the structure of ERalpha- and ERbeta-LBDs are examined. In addition, the findings from structural studies of ER-LBD in complex with peptide fragments corresponding to the NR-box II and III modules of the p160 coactivator TIF2 are discussed in the context of the assembly of ER:coactivator complexes. Together these studies have broadened our understanding of ER function by providing a unique insight into ERs ligand specificity, its ability to interact with coactivators and the structural changes that underlie receptor agonism and antagonism.

Twenty years on: the impact of fragments on drug discovery

After 20 years of sometimes quiet growth, fragment-based drug discovery (FBDD) has become mainstream. More than 30 drug candidates derived from fragments have entered the clinic, with two approved and several more in advanced trials. FBDD has been widely applied in both academia and industry, as evidenced by the large number of papers from universities, non-profit research institutions, biotechnology companies and pharmaceutical companies. Moreover, FBDD draws on a diverse range of disciplines, from biochemistry and biophysics to computational and medicinal chemistry. As the promise of FBDD strategies becomes increasingly realized, now is an opportune time to draw lessons and point the way to the future. This Review briefly discusses how to design fragment libraries, how to select screening techniques and how to make the most of information gleaned from them. It also shows how concepts from FBDD have permeated and enhanced drug discovery efforts.

Combining Hit Identification Strategies: Fragment- Based and in Silico Approaches to Orally Active 2-Aminothieno[2,3-D]Pyrimidine Inhibitors of the Hsp90 Molecular Chaperone.

Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential molecular therapeutic agents for the treatment of cancer. Here we describe novel 2-aminothieno[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors, which were designed by combining structural elements of distinct low affinity hits generated from fragment-based and in silico screening exercises in concert with structural information from X-ray protein crystallography. Examples from this series have high affinity (IC50 = 50-100 nM) for Hsp90 as measured in a fluorescence polarization (FP) competitive binding assay and are active in human cancer cell lines where they inhibit cell proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Several examples (34a, 34d and 34i) caused tumor growth regression at well tolerated doses when administered orally in a human BT474 human breast cancer xenograft model.

Towards predictive ligand design with free-energy based computational methods?

The accurate prediction of ligand-biopolymer binding affinities is of general interest to medicinal chemistry, as well as to the broader field of molecular recognition. The ability to predict computationally the thermodynamics of these molecular recognition processes has been relatively weak until recently, however, continued developments on several fronts are extending the scope of applicability of these methods. The rapid growth in the number of protein-ligand structures has initially led to the development of a range of empirical scoring functions based on relatively simple descriptions of intermolecular interactions. These methods have had some success in ranking binding affinities when tuned to particular protein systems or in rather qualitative estimates of molecular fit in fast docking calculations. However, they are too unreliable for more detailed, quantitative, assessment and comparison of binding affinities. Physics-based free energy calculations are in principle more general and have the potential to be significantly more accurate. These approaches have seen steady development over many years and rely on carefully calibrated molecular energy functions (force-fields), simulations of the systems with explicit solvent, and the coming-of-age of continuum solvation models. In addition to the initially developped Free Energy Perturbation (FEP) and Thermodynamic Integration (TI) methods, new approaches include the Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) and the Linear Interaction Energy (LIE) approaches. This review concentrates on MM-PBSA and LIE, and their variants. The routine application of these calculations is becoming possible because of enhanced computational hardware and the development of a range of computational chemistry tools. This review addresses: i) the basic principles behind free energy calculations ii) recent methodological advances iii) comparisons of predicted and experimentally determined affinities iv) the uncertainties and limitations of both the computational and experimental data v) areas where progress can be made vi) the practicality of applying the methods at the different stages of the drug discovery and optimization process.

Heat-stable antifreeze protein from grass.

We have discovered an antifreeze protein in an overwintering perennial ryegrass, Lolium perenne. The protein is stable at 100 °C and although it is a less effective antifreeze than proteins found in antarctic fish and insects, it is better at preventing ice recrystallization. This property enables grasses to tolerate ice formation in their tissues without being damaged, suggesting that the control of ice-crystal growth rather than the prevention of freezing may have evolved to be the critical factor in their survival at very low temperatures.

Design and characterization of libraries of molecular fragments for use in NMR screening against protein targets

We have designed four generations of a low molecular weight fragment library for use in NMR-based screening against protein targets. The library initially contained 723 fragments which were selected manually from the Available Chemicals Directory. A series of in silico filters and property calculations were developed to automate the selection process, allowing a larger database of 1.79 M available compounds to be searched for a further 357 compounds that were added to the library. A kinase binding pharmacophore was then derived to select 174 kinase-focused fragments. Finally, an additional 61 fragments were selected to increase the number of different pharmacophores represented within the library. All of the fragments added to the library passed quality checks to ensure they were suitable for the screening protocol, with appropriate solubility, purity, chemical stability, and unambiguous NMR spectrum. The successive generations of libraries have been characterized through analysis of structural properties (molecular weight, lipophilicity, polar surface area, number of rotatable bonds, and hydrogen-bonding potential) and by analyzing their pharmacophoric complexity. These calculations have been used to compare the fragment libraries with a drug-like reference set of compounds and a set of molecules that bind to protein active sites. In addition, an analysis of the overall results of screening the library against the ATP binding site of two protein targets (HSP90 and CDK2) reveals different patterns of fragment binding, demonstrating that the approach can find selective compounds that discriminate between related binding sites.