A non‐covalent inhibitor XMU‐MP‐3 overrides ibrutinib‐resistant Btk^C481S mutation in B‐cell malignancies

Abstract

Background and Purpose: Bruton's tyrosine kinase (BTK) plays a key role in B-cell receptor signalling by regulating cell proliferation and survival in various B-cell malignancies. Covalent low-MW BTK kinase inhibitors have shown impressive clinical efficacy in B-cell malignancies. However, the mutant Btk C481S poses a major challenge in the management of B-cell malignancies by disrupting the formation of the covalent bond between BTK and irreversible inhibitors, such as ibrutinib. The present studies were designed to develop novel BTK inhibitors targeting ibrutinib-resistant Btk C481S mutation. Experimental Approach: BTK-Ba/F3, BTK(C481S)-Ba/F3 cells, and human malignant B-cells JeKo-1, Ramos, and NALM-6 were used to evaluate cellular potency of BTK inhibitors. The in vitro pharmacological efficacy and compound selectivity were assayed via cell viability, colony formation, and BTK-mediated signalling. A tumour xenograft model with BTK-Ba/F3, Ramos and BTK(C481S)-Ba/F3 cells in Nu/nu BALB/c mice was used to assess in vivo efficacy of XMU-MP-3. Key Results: XMU-MP-3 is one of a group of low MW compounds that are potent non-covalent BTK inhibitors. XMU-MP-3 inhibited both BTK and the acquired mutant BTKC481S, in vitro and in vivo. Further computational modelling, sitedirected mutagenesis analysis, and structure-activity relationships studies indicated that XMU-MP-3 displayed a typical Type-II inhibitor binding mode. Conclusion and Implications: XMU-MP-3 directly targets the BTK signalling pathway in B-cell lymphoma. These findings establish XMU-MP-3 as a novel inhibitor of BTK, which could serve as both a tool compound and a lead for further drug development in BTK relevant B-cell malignancies, especially those with the acquired ibrutinibresistant C481S mutation.

Innovative Therapeutic Approaches in Primary Cutaneous B Cell Lymphoma

“…In BTK C481S the covalent bond between BTK and the inhibitor is disrupted, which results in reduced drug efficacy. XMU-MP-3 is a lowmolecular-weight, non-covalent BTK inhibitor, which has been shown to bind and inhibit both BTK and BTK C481S in vitro and in vivo (47). So far, no clinical trials have been initiated.…”

Targeting Bruton tyrosine kinase using non-covalent inhibitors in B cell malignancies

“…In addition to the several non-covalent BTK inhibitors that have shown promising efficacy in clinical trials, there are also some other non-covalent BTK inhibitors with significant antitumor effects in preclinical studies (Table 4 ). For instance, XMU-MP-3, a non-covalent inhibitor with potent BTK inhibitory activity, inhibited B cell lymphoma cells with or without BTK C481S mutation in vitro and in vivo [ 65 ], suggesting it could be effective in treating B cell lymphomas including those resistant to ibrutinib. Several other non-covalent BTK inhibitors including CB1763, GNE-431, and CGI-1746 have shown potent inhibitory effects on both wildtype and C481S mutant BTK [ 66 , 67 ].…”

“…XMU-MP-3 suppresses the proliferation of BTK-transformed Ba/F3 cell with an IC50 of 11.4 nM. It remains active against C481S mutant BTK-transformed Ba/F3 cells with an IC50 of 182.3 nM XMU-MP-3 remarkably inhibits tumor growth in BTK-transformed Ba/F3 and Ramos in mouse xenograft models without affecting animal weights [ 65 ] CB1763 (also known as AS-1763) NA NA CB1763 potently, reversibly inhibits both WT and C481S mutant BTKs (IC50 = 0.85 and 0.99 nM for WT and C481S, respectively). CB1763 substantially reduces BTK Tyr223 autophosphorylation at nanomolar concentration in HEK293 cells that are transfected with C481S mutant BTK CB1763 shows excellent antitumor activity in the BTK-driven OCI-Ly10 xenograft model [ 92 ] GNE-431 C 30 H 32 N 10 O 2 GNE-431 potently inhibits WT BTK and C481S mutant BTK (IC50 = 3.2 and 2.5 nM for WT and C481S mutant, respectively).…”

Non-Covalent BTK Inhibitors—The New BTKids on the Block for B-Cell Malignancies

“…As well as those described above with clinical trial data, there are a number of non-covalent BTKi in preclinical development. XMU-MP-3 [ 68 ], CB1763 (AS-1763) [ 69 ] and GNE-431 [ 70 ] all have in vitro activity against both wild type and C481 mutant BTK. Additionally, XMU-MP-3 and CB1763 have significant antitumour activity in vivo in xenograft models [ 68 , 69 ].…”

“…XMU-MP-3 [ 68 ], CB1763 (AS-1763) [ 69 ] and GNE-431 [ 70 ] all have in vitro activity against both wild type and C481 mutant BTK. Additionally, XMU-MP-3 and CB1763 have significant antitumour activity in vivo in xenograft models [ 68 , 69 ]. Another agent CGI-1746 inhibits BTK by stabilising the inactive form [ 71 ], but no publically available in vivo data are available at the time of writing.…”