Periodic Reporting for period 1 - LDC4PCaTher (BMX-targeted ligand-drug conjugates for prostate cancer therapy) Reporting period: 2016-11-01 to 2018-10-31 Summary of the context and overall objectives of the project Cancer is the second leading cause of death worldwide with estimated 18.1 million new cases in 2018 that resulted in 9.6 million deaths, a number that is expected to grow within the next decades. One of the most frequently diagnosed types of cancer is Prostate Cancer (PCa), with 1.28 million cases reported worldwide in 2018. Despite the advances in chemotherapy, the risks associated with current therapies still carry severe side effects and offer limited benefit to the patients. These side effects arise from the fact that cytotoxic agents that should preferentially kill cancer cells and not healthy tissues often display poor targeting performances and accumulate in normal healthy cells as well as in tumours. As a consequence, the risks and side effects associated with current therapies range from erectile dysfunction, loss of fertility, and incontinence after surgery or even recurrence of an androgen-independent tumour after androgen-ablation therapy. The need for novel safe and effective treatments is therefore urgent.During this project, we aim to develop new ligand–drug conjugates (LDCs) that can efficiently target prostate cancer cells and delivered the cytotoxic payload in the tumour, killing the cancer cells and sparing the healthy ones. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far During the period covered by this report, the majority of the objectives were fully achieved. We prepared a library of BMX inhibitors that was characterized from a chemical, biochemical and biophysical perspective. A synthetic path was devise to obtain the library of analogues, envisioning substituents in pre-determined regions of the tool compound scaffold. These molecules were chemically characterized by 1H NMR, High Resolution Mass Spectrometry and final compounds’ purity further assessed by HPLC. These were further profiled through the in silico determination of physicochemical parameters like cLogP and LogS, and also in vitro particle size determination (using Dynamic Light Scattering) and permeability (using Parallel Artificial Membrane Permeability). All the molecules were evaluated for affinity against the target protein BMX and an X-ray crystal structure of the protein covalently linked to an inhibitor was obtained. The most potent compound of the series was further tested against a set of 36 BMX-related kinases for selectivity. The lead molecules identified in the first stage of the project were then tested in their capacity to inhibit proliferation of prostate cancer cell lines LNCaP and PC-3. The ligand-drug conjugate is currently being synthesised and will be evaluated in vivo after the period covered by the Fellowship.During this project, we were able to identify a family of potent compounds with optimized physicochemical properties, selective for the TEC family of kinases. This work is currently in the process of being patented to allow further commercial exploitation. Once the IP is secured, it will be published in high ranking peer reviewed journals and presented at international conferences. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Over the last years, targeted therapies have been changing the paradigm of the drug discovery in the pharmaceutical industry. The development of antibody drug conjugates (ADCs) that was heavily pursued in recent years, is now being replaced by alternative therapeutic approaches, mainly due to ADCs main limitations, such as the risk of immunogeneicity, limited tumour penetration and expensive development associated costs. At the onset of this project, ligand-drug conjugates (LDCs) were emerging as a promising alternative to overcome these limitations and their versatility also allowed the development of conjugates targeting a myriad of different targets. With this project, we aimed to develop the first intracellular targeted LDC, adding another layer of selectivity to the first generation LDCs. We expect, upon the completion of the project, to have developed a conjugated displaying superior performance in comparison to small molecules currently used in chemotherapy and existing LDCs. Furthermore, we found strong evidence of the beneficial effects of using combination therapy approaches and therefore, once completed, our studies will also open new avenues for the use of LDCs and BMX inhibitors as novel therapeutic options for the treatment of prostate cancer.