New Mechanism Based Approaches For Treating Prostate Cancer

Prostate cancer (PC) is generally dependent on the androgen signaling axis for tumor growth. PC is managed by androgen deprivation therapy (ADT). The tumors then frequently progress by restoring ADT-resistant AR signaling through mechanisms such as intratumoral androgen synthesis, overexpression of AR, expression of splice variants of AR and alteration in the balance of AR co-regulators. This stage of progression is termed castrate recurrent prostate cancer (CRPC). Moreover, ADT has many major undesirable acute and chronic side effects on various normal tissues. Therefore a more strategic therapy approach is one that would disrupt a functional arm of AR signaling critical for PC/CRPC growth but not for the essential physiological roles of AR in normal adult tissues. This thesis describes two different mechanism-based approaches to develop small molecule drugs that address the above problems. The transcription factor ELK1 tethers the androgen receptor (AR) to chromatin, enabling sustained activation of genes critical for growth in prostate cancer cell lines. The N-terminal A/B domain of AR [AR(A/B)], which excludes the ligand binding pocket of AR, is adequate for interaction with ELK1. This is significant because the major splice variants of AR (AR-V7) that lack the ligand binding domain, as well as overexpressed full length AR, are known to strongly support growth of castration resistant prostate cancer (CRPC). In our first approach to develop small molecule drugs for prostate cancer, we showed that both wtAR and AR-V7 synergize with ELK1 by coopting the two ERK docking sites on ELK1, independent of the classical mechanism of (transient) activation of ELK1 via phosphorylation by ERK. As the association of ELK1 and AR is only required for prostate tumor growth, disrupting this interaction should selectively inhibit the growth of CRPC cells without interfering with the physiological role of androgen in normal tissues. Therefore, small molecules that disrupt binding of AR to ELK1 should inhibit the growth of a broader spectrum of advanced prostate tumors than androgen ablation or conventional anti-androgen therapies without the many acute and chronic side effects associated with those treatments. We have developed and conducted a stringent in situ high throughput screen for small molecules that selectively disrupt the ELK1-AR synergy. We initially screened over 18,000 compounds from diversity sets that follow the Lipinski guidelines for “drug-likeliness”. Our top hit from the screen inhibited ELK1-dependent promoter activation by androgen in a dose-dependent manner but did not inhibit promoter activation via canonical androgen response elements. Follow up structure-activity studies identified a lead compound that was much more stable than the initial hit. We report discovery of this small molecule (KCI807) that selectively disrupts ELK1-dependent promoter activation by wild-type and variant forms of AR without interfering with ELK1 activation by ERK. KCI807 has an obligatory flavone scaffold and functional hydroxyl groups on C5 and C3\u27. KCI807 binds to purified AR, blocking ELK1 binding, and selectively blocks recruitment of AR to chromatin by ELK1. KCI807 narrowly affects a subset AR target growth genes and selectively inhibits AR-dependent growth of PCa cell lines and Enzalutamide-resistant PCa tumor xenografts. The results offer a mechanism-based therapeutic paradigm for disrupting the AR growth signaling axis in the spectrum of prostate tumors while avoiding global attenuation of testosterone actions. The second approach to developing new small molecule drugs against prostate cancer involved the development of hybrid molecules. Histone deacetylase inhibitors (HDACis) can disrupt androgen signaling through the down regulation of heat shock protein 90 (HSP90). However despite their ineffectiveness in prostate cancer (PCa) cells non-selective toxicities are associated with these molecules. We designed hybrid molecules containing partial scaffolds of the AR targeted drug, enzalutamide, and the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), to weaken the intrinsic pan-HDACi activity of the molecule and to selectively target the cytosolic AR-HSP90 complex in AR overexpressing and enzalutamide-resistant PCa cells. These new molecules, 2-75 and 1005, showed reduced potency in intrinsic HDAC inhibitor activity, degraded the HSP90 chaperone protein, induced hyper acetylation of the HDAC6 substrate α tubulin, induced p21, and caused loss of viability of the enz-resistant C4-2 cells all to a greater extent compared to either parent compound alone. These results suggested that these new molecules could be used as prototypes for the development of hybrid HDAC inhibiting drugs with reduced pan HDAC inhibitor activity and increased selectivity for AR overexpressing PC cells

Identification of ELK1 interacting peptide segments in the androgen receptor

Prostate cancer (PCa) growth requires tethering of the androgen receptor (AR) to chromatin by the ETS domain transcription factor ELK1 to coactivate critical cell proliferation genes. Disruption of the ELK1–AR complex is a validated potential means of therapeutic intervention in PCa. AR associates with ELK1 by coopting its two ERK docking sites, through the amino-terminal domain (A/B domain) of AR. Using a mammalian two-hybrid assay, we have now functionally mapped amino acids within the peptide segments 358–457 and 514–557 in the A/B domain as required for association with ELK1. The mapping data were validated by GST (glutathione S-transferase)-pulldown and BRET (bioluminescence resonance energy transfer) assays. Comparison of the relative contributions of the interacting motifs/segments in ELK1 and AR to coactivation of ELK1 by AR suggested a parallel mode of binding of AR and ELK1 polypeptides. Growth of PCa cells was partially inhibited by deletion of the upstream segment in AR and nearly fully inhibited by deletion of the downstream segment. Our studies have identified two peptide segments in AR that mediate the functional association of AR with its two docking sites in ELK1. Identification of the ELK1 recognition sites in AR should enable further structural studies of the ELK1–AR interaction and rational design of small molecule drugs to disrupt this interaction

Strategy for tumor selective disruption of androgen receptor function in the spectrum of prostate cancer

Purpose: Testosterone suppression in prostate cancer (PC) is limited by serious side effects and resistance via restoration of androgen receptor (AR) functionality. ELK1 is required for ARdependent growth in various hormone-dependent and castration resistant PC models. The amino terminal domain of AR docks at two sites on ELK1 to co-activate essential growth genes. This study explores the ability of small molecules to disrupt the ELK1-AR interaction in the spectrum of PC, inhibiting AR activity in a manner that would predict functional tumor selectivity. Experimental design: Small molecule drug discovery and extensive biological characterization of a lead compound. Results: We have discovered a lead molecule (KCI807) that selectively disrupts ELK1-dependent promoter activation by wild-type and variant ARs without interfering with ELK1 activation by ERK. KCI807 has an obligatory flavone scaffold and functional hydroxyl groups on C5 and C3'. KCI807 binds to AR, blocking ELK1 binding, and selectively blocks recruitment of AR to chromatin by ELK1. KCI807 primarily affects a subset of AR target growth genes selectively suppressing AR-dependent growth of PC cell lines with a better inhibitory profile than enzalutamide. KCI807 also inhibits in vivo growth of castration/enzalutamide-resistant cell line-derived and patient-derived tumor xenografts. In the rodent model, KCI807 has a plasma half-life of 6h and maintenance of its antitumor effect is limited by self-induced metabolism at its 3'-hydroxyl. Conclusions: The results offer a mechanism-based therapeutic paradigm for disrupting the AR growth-promoting axis in the spectrum of prostate tumors while reducing global suppression of testosterone actions. KCI807 offers a good lead molecule for drug development

Determinants of Bacteriophage 933W Repressor DNA Binding Specificity

We reported previously that 933W repressor apparently does not cooperatively bind to adjacent sites on DNA and that the relative affinities of 933W repressor for its operators differ significantly from that of any other lambdoid bacteriophage. These findings indicate that the operational details of the lysis-lysogeny switch of bacteriophage 933W are unique among lambdoid bacteriophages. Since the functioning of the lysis-lysogeny switch in 933W bacteriophage uniquely and solely depends on the order of preference of 933W repressor for its operators, we examined the details of how 933W repressor recognizes its DNA sites. To identify the specificity determinants, we first created a molecular model of the 933W repressor-DNA complex and tested the predicted protein-DNA interactions. These results of these studies provide a picture of how 933W repressor recognizes its DNA sites. We also show that, opposite of what is normally observed for lambdoid phages, 933W operator sequences have evolved in such a way that the presence of the most commonly found base sequences at particular operator positions serves to decrease, rather than increase, the affinity of the protein for the site. This finding cautions against assuming that a consensus sequence derived from sequence analysis defines the optimal, highest affinity DNA binding site for a protein

GrassPlot - a database of multi-scale plant diversity in Palaearctic grasslands

GrassPlot is a collaborative vegetation-plot database organised by the Eurasian Dry Grassland Group (EDGG) and listed in the Global Index of Vegetation-Plot Databases (GIVD ID EU-00-003). GrassPlot collects plot records (releves) from grasslands and other open habitats of the Palaearctic biogeographic realm. It focuses on precisely delimited plots of eight standard grain sizes (0.0001; 0.001;... 1,000 m(2)) and on nested-plot series with at least four different grain sizes. The usage of GrassPlot is regulated through Bylaws that intend to balance the interests of data contributors and data users. The current version (v. 1.00) contains data for approximately 170,000 plots of different sizes and 2,800 nested-plot series. The key components are richness data and metadata. However, most included datasets also encompass compositional data. About 14,000 plots have near-complete records of terricolous bryophytes and lichens in addition to vascular plants. At present, GrassPlot contains data from 36 countries throughout the Palaearctic, spread across elevational gradients and major grassland types. GrassPlot with its multi-scale and multi-taxon focus complements the larger international vegetationplot databases, such as the European Vegetation Archive (EVA) and the global database " sPlot". Its main aim is to facilitate studies on the scale-and taxon-dependency of biodiversity patterns and drivers along macroecological gradients. GrassPlot is a dynamic database and will expand through new data collection coordinated by the elected Governing Board. We invite researchers with suitable data to join GrassPlot. Researchers with project ideas addressable with GrassPlot data are welcome to submit proposals to the Governing Board

Affinities of wild-type 933W repressor protein for synthetic 933W operators.

<p>The affinities of 933W repressor for the substituted DNAs are expressed as the dissociation constants of these operators for 933W repressor. NS-DNA that does not contain a DNA sequence to which 933W repressor binds specifically. The affinity of 933W repressor for non-specific DNA is give as an intrinsic ‘per-site’ dissociation constant (K_D^NS = K_D^OBS[2L-n], where L is DNA length and n is site size <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034563#pone.0034563-JenJacobson1" target="_blank">[28]</a>). The protein concentrations used were corrected for activity.</p

Naturally occurring 933W operator site sequences.

<p><i>(A)</i> The sequences and affinity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034563#pone.0034563-Bullwinkle1" target="_blank">[12]</a> (dissociation constants, K_D) of 933W repressor binding for naturally occurring 933W binding sites. <i>(B)</i> Logo sequence alignment of the ten ‘half-sites’ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034563#pone.0034563-Schneider1" target="_blank">[20]</a> found within the five repressor operator sites and the consensus 933W repressor binding site sequence constructed from <i>in vitro</i> selection <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034563#pone.0034563-Koudelka1" target="_blank">[11]</a>.</p

Model of the 933W repressor-operator complex.

<p><i>(A)</i> 933W repressor in complex with the 933W consensus DNA site. The complex is oriented looking down the C-terminal portion of the DNA contacting (“recognition") to the N-terminal portions. The identity of the putative DNA contacting residues is indicated and highlighted in purple. The DNA is modeled on the right with the “top" strand (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034563#pone-0034563-g001" target="_blank">Figure 1C</a>) shown in grey and the complementary strand in black. <i>(B)</i> Schematic representation of the contacts identified from the model built structure between residues in the recognition helix of 933W repressor and bases in the 933W consensus half site DNA sequence. Predicted interactions are depicted by arrows.</p

Role of Position 4 base substituents in determining 933W repressor affinity for DNA.

<p>(A) The base sequence at position 4 in one half site of the 933W binding site was changed as indicated. Dissociation constants were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034563#s2" target="_blank">Methods and Materials</a>. (B) DNase I footprinting analysis of complexes between 933W repressor and 933W consensus sequence or its position 4 variants shown in (A) were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034563#s2" target="_blank">Methods and Materials</a>. Shown is a Phosphoromager scan of the gel. The sequence of the operators are shown together with the positions of the bands resulting from DNase I cleavages in the absence (−) and presence (+) of saturating concentrations of 933W repressor.</p