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Targeting cancer-related proteins : rational design of covalent inhibitors and degraders for TEC kinases and serine hydrolases
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Targeted therapy has significantly transformed the landscape of cancer treatment by providing several advantages in contrast to conventional chemotherapy. Small covalent inhibitors and proteolysis targeting chimeras (PROTACs) are designed to specifically target disease-causing proteins in malignant cells, while leaving the healthy cells unaffected. This specificity makes these therapies more effective and reduces the risk of adverse side effects. Adding to the vast list of proteins amenable to targeting via these chemical routes, Bruton’s tyrosine kinase (BTK) and acyl-protein thioesterase 1 (APT1), have shown therapeutic promise for the treatment of haematological cancers and other related diseases. Within this research, we employed a rational approach to develop covalent small molecules that selectively bind to BTK, and PROTACs for APT1. We subsequently evaluated their efficacy in treating blood cancers. We first investigated the therapeutic potential of a small covalent molecule (JS25) with nanomolar activity against BTK. JS25 was obtained from the scaffold of BMX-IN-1 (a recently discovered BTK inhibitor), as part of our efforts to identify regions of the molecule that could be modulated for improved efficacy and selectivity. We sought to characterize the binding mode of JS25 to BTK and asserted its selectivity against a panel of proteins related to BTK’s signalling pathway or with an equally placed cysteine as to the cysteine 481 of BTK. Validation of its therapeutic effect was conducted using xenograft mice models of Burkitt’s lymphoma, and patient-derived models of diffuse large B-cell lymphoma and chronic lymphocytic leukaemia. Finally, we explored the capability of JS25 to cross the brain−blood barrier and treat infiltration of tumour cells in the brain. JS25 presented a high binding efficacy to BTK and with a more desirable selectivity and inhibitory profile compared to the clinically approved BTK inhibitors Ibrutinib and Acalabrutinib. Structural prediction of the BTK/JS25 complex revealed sequestration of tyrosine 551 responsible for rendering BTK to its inactive state. JS25 also inhibited the proliferation of myeloid and B-lymphoid cancer cell lines. In preclinical models of B-cell cancers, JS25 treatment induced a more pronounced cell death in a murine xenograft model of Burkitt’s lymphoma, causing a 30–40% reduction of the subcutaneous tumour and an overall reduction in the percentage of metastasis and secondary tumour formation. In a patient model of diffuse large B-cell lymphoma, the drug response of JS25 was higher than that of Ibrutinib, leading to a 64% on-target efficacy. Finally, in zebrafish patient-derived xenografts of chronic lymphocytic leukaemia, JS25 was faster and more effective in decreasing tumour burden, producing superior therapeutic effects compared to Ibrutinib. In the second part, we integrated computational and biochemical platforms to help accelerate the identification of novel PROTACs with improved selectivity for APT1. PRosettaC protocol was utilized to predict the optimal linker length, configuration, and binding efficacy of designed PROTACs to APT1 and E3 ubiquitin ligase. Activity-based protein profiling and immunoblot assays were employed to monitor the inhibition and degradation of APT1 and to assert their selectivity. Additionally, the anti-tumour activity of the most promising APT1-targeting PROTAC was assessed in blood cancer cell lines. In accordance with the computer modelling, PROTACs with shorter linker lengths had limited or minimal effectiveness in degrading APT1. Conversely, PROTACs that comprehended linkers with long alkyl chains exhibited potent degradation in the nanomolar range, as these mimic the structure of substrates specific for this enzyme. Of particular interest is the PROTAC C8, which exhibited a high selectivity for APT1 and activity in several blood cancer cell lines. Notably, C8 displays remarkable selectivity for APT1, without causing significant disruption of other serine hydrolases, such as ABHDs, which are often the targets of APT inhibitors. Overall, we endeavoured to harness the advantages of computational modelling and preclinical validation to create novel targeted therapeutic agents that could offer superior efficacy, selectivity, and safety profiles. It is hoped that the findings and efforts here presented will make a significant contribution to the expanding field of targeted therapy and pave the way for the development of innovative treatments for haematological cancers and other ailments with unmet clinical treatments.
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terapia-alvo inibidores covalentes PROTACs cancros hematológicos targeted therapy covalent inhibitors haematological cancers