A Small Molecule Drug Conjugate SMDC for Prostate Cancer Therapy

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A scalable insect cell-based production process of the human recombinant BMX for in-vitro covalent ligand high-throughput screening

Publication . Sousa, Bárbara B.; Sousa, Marcos F. Q.; Marques, Marta C.; Seixas, João D.; Brito, José A.; Matias, Pedro M.; Bernardes, Gonçalo J. L.; Roldão, António

Bone Marrow Tyrosine kinase in the chromosome X (BMX) is a TEC family kinase associated with numerous pathological pathways in cancer cells. Covalent inhibition of BMX activity holds promise as a therapeutic approach against cancer. To screen for potent and selective covalent BMX inhibitors, large quantities of highly pure BMX are normally required which is challenging with the currently available production and purification processes. Here, we developed a scalable production process for the human recombinant BMX (hrBMX) using the insect cell-baculovirus expression vector system. Comparable expression levels were obtained in small-scale shake flasks (13 mL) and in stirred-tank bioreactors (STB, 5 L). A two-step chromatographic-based process was implemented, reducing purification times by 75% when compared to traditional processes, while maintaining hrBMX stability. The final production yield was 24 mg of purified hrBMX per litter of cell culture, with a purity of > 99%. Product quality was assessed and confirmed through a series of biochemical and biophysical assays, including circular dichroism and dynamic light scattering. Overall, the platform herein developed was capable of generating 100 mg purified hrBMX from 5 L STB in just 34 days, thus having the potential to assist in-vitro covalent ligand high-throughput screening for BMX activity inhibition.

2020Journal article

Restricted access

Structural and biophysical insights into the mode of covalent binding of rationally designed potent BMX inhibitors

Publication . Seixas, João D.; Sousa, Bárbara B.; Marques, Marta C.; Guerreiro, Ana; Traquete, Rui; Rodrigues, Tiago; Albuquerque, Inês S.; Sousa, Marcos F. Q.; Lemos, Ana R.; Sousa, Pedro M. F.; Bandeiras, Tiago M.; Wu, Di; Doyle, Shelby K.; Robinson, Carol V.; Koehler, Angela N.; Corzana, Francisco; Matias, Pedro M.; Bernardes, Gonçalo J. L.

The bone marrow tyrosine kinase in chromosome X (BMX) is pursued as a drug target because of its role in various pathophysiological processes. We designed BMX covalent inhibitors with single-digit nanomolar potency with unexploited topological pharmacophore patterns. Importantly, we reveal the first X-ray crystal structure of covalently inhibited BMX at Cys496, which displays key interactions with Lys445, responsible for hampering ATP catalysis and the DFG-out-like motif, typical of an inactive conformation. Molecular dynamic simulations also showed this interaction for two ligand/BMX complexes. Kinome selectivity profiling showed that the most potent compound is the strongest binder, displays intracellular target engagement in BMX-transfected cells with two-digit nanomolar inhibitory potency, and leads to BMX degradation PC3 in cells. The new inhibitors displayed anti-proliferative effects in androgen-receptor positive prostate cancer cells that where further increased when combined with known inhibitors of related signaling pathways, such as PI3K, AKT and Androgen Receptor. We expect these findings to guide development of new selective BMX therapeutic approaches.

2020Journal article

Open access