A Targeted Bivalent Androgen Receptor Binding Compound for Prostate Cancer Therapy.

The androgen-directed treatment of prostate cancer (PCa) is fraught with the recurrent profile of failed treatment due to drug resistance and must be addressed if we are to provide an effective therapeutic option. The most singular difficulty in the treatment of PCa is the failure to respond to classical androgen withdrawal or androgen blockade therapy, which often develops as the malignancy incurs genetic alterations and gain-of-function somatic mutations in the androgen receptor (AR). Physical cellular damaging therapeutic agents, such as radiation or activatable heat-generating transducers would circumvent classical "anti-functional" biological resistance, but to become ultimately effective would require directed application modalities. To this end, we have developed a novel AR-directed therapeutic agent by creating bivalent androgen hormone-AF-2 compounds that bind with high affinity to AR within cells. Here, we used molecular modeling and synthetic chemistry to create a number of compounds by conjugating 5α-dihydrotestosterone (DHT) to various AF-2 motif sequence peptides, through the use of a glycine and other spacer linkers. Our data indicates these compounds will bind to the AR in vitro and that altering the AF-2 peptide composition of the compound does indeed improve affinity for the AR. We also show that many of these bivalent compounds can readily pass through the plasma membrane and effectively compete against androgens alone.

Demonstration of a plasmonic thermocycler for the amplification of human androgen receptor DNA.

A plasmonic heating method for the polymerase chain reaction is demonstrated by the amplification of a section of the human androgen receptor gene. The thermocycler has a simple low-cost design, demonstrates excellent temperature stability and represents the first practical demonstration of plasmonic thermocycling.

Dynamic rewiring of the androgen receptor protein interaction network correlates with prostate cancer clinical outcomes.

The androgen receptor (AR) is a ligand-inducible transcription factor, a member of the nuclear receptor superfamily, which plays an important role in the development and progression of prostate cancer (CaP). The transformation to CaP has been linked to several somatic AR gene mutations and changes in AR protein complex formation, which in turn increase the potential activity of the receptor. Thus, to address the mechanism of AR-mediated neoplastic transformation, we developed in vitro methodology to isolate and characterize, via mass spectrometry, AR complexes of three AR genetic variants: wild type-AR, and two somatic gain-of-function AR prostatic mutants (T877A-AR and 0CAG-AR isoforms). To fully characterize the significance of our large raw data set, we employed a sophisticated systems biology approach to create an integrative protein-interaction network profile for each AR isoform. Our comparative analysis identified subnetwork cluster profiles for AR isoforms (WT, T877A, and 0CAG) that segregated AR isoforms on the basis of androgen stimulation conditions and mutant aggressiveness. Furthermore, results from additional correlative gene microarray analysis studies of all three AR isoform (WT, T877A, 0CAG) subnetwork clusters were assessed and found to be significantly enriched in tumor versus normal prostate tissues. We also identified two AR-interaction clusters, containing 21 and 30 proteins, respectively, that showed unfavourable prognosis outcome of recurrent cancers, on the basis of PSA, Gleason score and combined PSA/Gleason score. In conclusion, we have characterized a large panel of novel AR-interacting proteins, through a combined proteomics/systems biology screen, that are of clinical relevance and could potentially serve as novel markers for diagnosis and prognosis of CaP.

Handheld impedance biosensor system using engineered proteinaceous receptors.

We put forward an impedometric protein-based biosensor platform suitable for point-of-care diagnostics. A hand-held scale impedance reader system is described for the detection of corresponding physiochemical changes as the immobilized proteins bind to the analyte molecules in the proximity of the microfabricated electrodes. Specifically, we study the viability of this approach for glucose biosensing purposes using genetically engineered glucokinase as receptor proteins. The proposed reagent-less biosensor offers a high sensitivity of 0.5 mM glucose concentration level in the physiologically relevant range of 0.5 mM to 7.5 mM with less than 10 s response time.

An investigation into CAG repeat length variation and N/C terminal interactions in the T877A mutant androgen receptor found in prostate cancer.

Prostate cancer may progress by circumventing ablation therapy due to mutations in the androgen receptor (AR) gene. The most intensively studied is the T877A mutation in the ligand binding domain (LBD), which causes the AR to become promiscuous, i.e., respond to a number of different ligands. Our investigations have shown that the T877A mutation alters the inverse relationship between CAG repeat length and transactivation in a noticeable albeit minor manner, while increasing N/C terminal interactions. In the presence of beta-catenin, a coactivator over-expressed in prostate cancer, the inverse relationship between CAG repeat length and transactivation is reversed for the wild type (wt) AR as well. We have also used molecular modeling with the AR and FXXLF and LXXLL peptides to investigate N/C terminal and coactivator interactions. In T877A, this approach revealed an increase in the flexibility of amino acid residues in the activation function 2 (AF-2) domain in the LBD, and a larger solvent accessible surface in T877A compared to the wt AR AF-2 domain. Thus, the improved induced fit of the AR N-terminal domain FXXLF-containing peptide into the T877A LBD could be due to the increased flexibility and solvent accessibility of the AF-2 domain. These new observations suggest that the AR CAG effect can be overridden by prostate cancer mutations, and also further our understanding of hormone-refractory prostate cancer by helping to explain the promiscuity of the T877A mutation.

Impaired helix 12 dynamics due to proline 892 substitutions in the androgen receptor are associated with complete androgen insensitivity.

Structural studies of the ligand-binding domain (LBD) of several steroid receptors have revealed that the dynamic properties of the C-terminal helix 12 (H12) are the major determinant of the activation mode of these receptors. H12 exhibits high mobility and different conformations in the absence of ligand. Upon ligand binding, H12 is stabilized in a precise position to seal the ligand-binding pocket and finalize the assembly of the activation function (AF-2) domain. In this study, we investigated the role of the conserved proline 892 of the androgen receptor (AR) in directing the dynamic location and orientation of the AR-H12. We used a combined approach including kinetic and biochemical assays with molecular dynamic simulations to analyze two substitutions (P892A and P892L) identified in individuals with complete androgen insensitivity syndrome. Our analyses revealed distinct mechanisms by which these substitutions impair H12 function resulting in severely defective receptors. The AR-P892A receptor exhibited reduced ligand binding and transactivational potential because of an increased flexibility in H12. The AR-P892L substitution renders the receptor inactive due to a distorted, unstructured and misplaced H12. To confirm the mutants' inability to stabilize H12 in an active position, we have developed a novel in vivo assay to evaluate the accessibility of the H12-docking site on the AR-LBD surface. An extrinsic AR-H12 peptide was able to interact with wild-type and mutant LBDs in the absence of ligand. Ligand-induced proper positioning of the intrinsic H12 of wild-type AR prevented these interactions, whereas the misplacement of the mutants' H12 did not. Proline at this position may be critical for H12 dynamics not only in the AR, but also in other nuclear receptors where this proline is conserved.

The use of androgen receptor amino/carboxyl-terminal interaction assays to investigate androgen receptor gene mutations in subjects with varying degrees of androgen insensitivity.

Five mutations in the ligand-binding domain (LBD) of the human androgen receptor (hAR) found in patients with varying degrees of androgen insensitivity syndrome (AIS) were investigated for their effects on receptor dynamics. These were Arg(871)Gly (mild), Ser(814)Asn (partial), Glu(772)Ala (partial), Val(866)Met (complete), and Arg(774)Cys (complete). Previous analysis showed that the mutant receptors exhibited near-normal kinetics, except Arg(774)Cys, which had severely reduced androgen binding, and Val(866)Met, which showed increased equilibrium dissociation constant (K(d)) and elevated dissociation rate (k) values. Ser(814)Asn exhibited ligand-selective k values, i.e. increased for dihydrotestosterone and mibolerone, but normal for methyltrenolene. Using mammalian two-hybrid assays, hAR amino/carboxyl (N/C)-terminal interactions of the mutant receptors were analyzed in the presence and absence of the hAR coactivator transcription intermediary factor 2 (TIF2). The mutations conferred decreased hAR N/C-terminal interaction, i.e. mild (approximately 1.5-fold), partial (2-fold), and complete (10-fold), that mirrored the degree of AIS. All mutant LBDs showed a 2- to 3-fold increase in N/C-terminal interactions when TIF2 was cotransfected, although of a magnitude still less than that of wild-type LBD with TIF2. The ligand-selective properties of the Ser(814)Asn mutant were also clearly reflected by the N/C-terminal interactions. Thus, measurement of N/C-terminal interactions may assist in the molecular analysis of mutant hARs associated with AIS.