Identification of quinazoline-affine proteins in malignant cells

Quinazoline-based alpha1-adrenergic antagonists like prazosin or doxazosin are traditional drugs to treat hypertension or benign prostate hyperplasia (BHP). Recent studies in our lab showed that alpha1-adrenergic antagonists induce apoptosis as well as signs of differentiation in human erythroleukemia cell lines. Surprisingly, these effects were shown to be independent of the presence of alpha1-adrenoceptors on leukemia cells. Similar to leukemia cells, alpha1-adrenergic antagonists exert an alpha1-adrenoceptor independent pro-apoptotic effect on hyperplastic prostate cells as well as prostate cancer cells. Based on these novel pro-apoptotic effects of alpha1- adrenergic antagonists on cancer cells, these drugs are currently discussed for application in chemotherapy. In particular, the use of alpha1-adrenoceptors antagonists in the treatment of androgen independent prostate cancer is proposed, since this clinical manifestation of prostate cancer goes along with complications like tumour relapse and metastasis which are associated with poor prognosis. Unfortunately, the application of alpha1-adrenergic antagonists per se in cancer treatment seems to be risky because of the wide spectrum of expected (adrenergic!) effects of these drugs, in particular concerning blood pressure. Therefore, it is appropriate to identify the so far unidentified non-adrenoceptor-targets of alpha1-adrenergic antagonists in order to engineer new drugs with high affinity to this unknown target associated with induction of apoptosis, but with low affinity to alpha1- adrenoceptors. Using a fluorescent derivate of prazosin - BODIPY FL Prazosin - we were able to visualize the non-adrenergic target(s), recognized by alpha1-adrenergic drugs in human erythroleukemia cells. In the current project proposal we present a strategy how to enrich and to identify the non-adrenergic targets with affinity to adrenergic antagonists in erythroleukemia and prostate cancer cells, based on labelling with BODIPY FL Prazosin. Cancer cell proteins with affinity to BODIPY FL Prazosin will be purified and analyzed by functional proteomics. The function of potential target-proteins will be verified by experimental modulation of the expression in the cell models. The successful identification of these so far unidentified quinazoline sensitive targets will disclose a potential Achilles heel sensitive to therapeutic intervention in human malignancies and will lay the foundation for the engineering of highly specific drugs tailored for these targets. The broad range of possible application areas of these drugs include benign and malign alterations of the prostate as well as hematopoietic malignancies. Furthermore, the identification of these targets will gain new insights into the regulation of cell death as well as hematopoietic differentiation.

Quinazolines are a group of drugs, originally designed for the treatment of hypertension and benign hyperplasia of the prostate. Interestingly, it was found that these drugs are also able to inhibit the growth of malignant cells. However, the underlying mechanisms of these anti- tumour effects were only sparsely characterized as yet. The main finding of our project is that prazosin - a member of the drug class of quinazolines - enters the cell via a mechanism termed endocytosis and subsequently induces apoptosis, a cellular suicide program. Thus, our results imply, that prazosin binds to a protein localized at the cell surface holding an essential role in maintaining the survival of tumour cells. Furthermore, we demonstrated that prazosin induced cell death is functionally linked to a unique morphological reorganisation of the lysosomes which are the cellular digestion organelles. Distinct inhibitors of endocytosis were able to impede the reorganisation of the lysosomes and also restored the viability of cells in the presence of prazosin, implying a functional link between endocytosis, lysosomes and the induction of cell death. Further experiments showed that prazosin enters cells via so called lipid rafts, known as specialized regions in the cell membrane with a unique lipid composition. The final destination of the drug on its way through the cell is the lysosome compartment which subsequently changes its morphology from vesicular structures to tubular structures; a process referred to as lysosomal tubulation in the literature. Lysosomal tubules formed a complex network throughout the whole cell and were the origin of long polar needle-like protrusions. Herein, the lysosomal pH was shown to be an important factor. Raising the pH in the lysosomes completely abolished the lysosomal tubulation process and restored the viability of prazosin treated cells. In order to identify the main target protein of quinazolines, we employed a proteomics strategy based on using a fluorescent version of prazosin (QAPB) and mass spectrometry. Herein, we indeed discovered a protein fraction with affinity towards QAPB. Also a couple of membrane proteins were found in this protein fraction. Some of them were mainly expressed at the cell membrane in untreated cells, but appeared on tubular lysosomal structures in prazosin treated cells. However, a functional link between these proteins and prazosin still needs to be established. This will be subject of further studies. In summary, we have elucidated the uptake, the intracellular trafficking and the main target organelle of prazosin the lysosomes. Thus, we discovered fundamental mechanisms of the pro-apoptotic activity of quinazolines and laid the basis for the identification of the main target protein. Therefore, our project contributed significantly to the ongoing initiative to exploit the promising anti-tumour effects of quinazolines for future use in chemotherapeutic regimes for the treatment of cancer.