Tan, V. M. | bioRxiv Science

bioRxiv2023

Saracatinib synergizes with enzalutamide to downregulate androgen receptor activity in castration resistant prostate cancer

Prostate cancer (PCa) remains the most diagnosed non-skin cancer amongst the American male population. Treatment for localized prostate cancer consists of androgen deprivation therapies (ADTs), which typically inhibit androgen production and the androgen receptor (AR). Though initially effective, a subset of patients will develop resistance to ADTs and the tumors will transition to castration-resistant prostate cancer (CRPC). Second generation hormonal therapies such as abiraterone acetate and enzalutamide are typically given to men with CRPC. However, these treatments are not curative and typically prolong survival only by a few months. Several resistance mechanisms contribute to this lack of efficacy such as the emergence of AR mutations, AR amplification, lineage plasticity, AR splice variants (AR-Vs) and increased kinase signaling. Having identified SRC kinase as a key tyrosine kinase enriched in CRPC patient tumors from our previous work, we evaluated whether inhibition of SRC kinase synergizes with enzalutamide or chemotherapy in several prostate cancer cell lines expressing variable AR isoforms. We observed robust synergy between the SRC kinase inhibitor, saracatinib, and enzalutamide, in the AR-FL+/AR-V+ CRPC cell lines, LNCaP95 and 22Rv1. We also observed that saracatinib significantly decreases AR Y534 phosphorylation, a key SRC kinase substrate residue, on AR-FL and AR-Vs, along with the AR regulome, supporting key mechanisms of synergy with enzalutamide. Lastly, we also found that the saracatinib-enzalutamide combination reduced DNA replication compared to the saracatinib-docetaxel combination, resulting in marked increased apoptosis. By elucidating this combination strategy, we provide pre-clinical data that suggests combining SRC kinase inhibitors with enzalutamide in select patients that express both AR-FL and AR-Vs.

cancer biology↗

bioRxiv2021

Massively parallel, computationally-guided design of a pro-enzyme

Confining the activity of a designed protein to a specific microenvironment would have broad-ranging applications, such as enabling cell type-specific therapeutic action by enzymes while avoiding off-target effects. While many natural enzymes are synthesized as inactive zymogens that can be activated by proteolysis, it has been challenging to re-design any chosen enzyme to be similarly stimulus-responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for pro-enzyme design. As a model system, we employ carboxypeptidase G2 (CPG2), a clinically approved enzyme that has applications in both the treatment of cancer and controlling drug toxicity. Detailed kinetic characterization of the most effective designed variants shows that they are inhibited by approximately 80% compared to the unmodified protein, and their activity is fully restored following incubation with site-specific proteases. Introducing disulfide bonds between the pro-and catalytic domains based on the design models increases the degree of inhibition to 98%, but decreases the degree of restoration of activity by proteolysis. A selected disulfide-containing pro-enzyme exhibits significantly lower activity relative to the fully activated enzyme when evaluated in cell culture. Structural and thermodynamic characterization provides detailed insights into the pro-domain binding and inhibition mechanisms. The described methodology is general and could enable the design of a variety of pro-proteins with precise spatial regulation. SignificanceProteins have shown promise as therapeutics and diagnostics, but their effectiveness is limited by our inability to spatially target their activity. To overcome this limitation, we developed a computationally-guided method to design inactive "pro-enzymes" or "zymogens," which are activated through cleavage by a protease. Since proteases are differentially expressed in various tissues and disease states, including cancer, these pro-enzymes could be targeted to the desired microenvironment. We tested our method on the therapeutically-relevant protein, carboxypeptidase G2 (CPG2). We designed Pro-CPG2s that are inhibited by 80-98% and are partially to fully re-activatable following protease treatment. The developed methodology, with further refinements, could pave the way for routinely designing protease-activated protein-based therapeutics and diagnostics that act in a spatially controlled manner.

biophysics↗