Structural basis for small molecule targeting of Doublecortin Like Kinase 1 with DCLK1-IN-1

Abstract

Doublecortin-like kinase 1 (DCLK1) is an understudied bi-functional kinase with a proven role in tumour growth and development. However, the presence of tissue-specific spliced DCLK1 isoforms with distinct biological functions have challenged the development of effective strategies to understand the role of DCLK1 in oncogenesis. Recently, DCLK1-IN-1 was reported as a highly selective DCLK1 inhibitor, a powerful tool to dissect DCLK1 biological functions. Here, we report the crystal structures of DCLK1 kinase domain in complex with DCLK1-IN-1 and its precursors. Combined, our data rationalises the structure-activity relationship that informed the development of DCLK1-IN-1 and provides the basis for the high selectivity of DCLK1-IN-1, with DCLK1-IN-1 inducing a drastic conformational change of the ATP binding site. We demonstrate that DCLK1-IN-1 binds DCLK1 long isoforms but does not prevent DCLK1’s Microtubule-Associated Protein (MAP) function. Together, our work provides an invaluable structural platform to further the design of isoform-specific DCLK1 modulators for therapeutic intervention.

Doublecortin‐like kinase 1 is a therapeutic target in squamous cell carcinoma

“…Currently there is a DCLK1 inhibitor, DCLK1‐IN‐1, which has been reported to inhibit DCLK1 activity with high specificity 32 . Nevertheless, DCLK1‐IN‐1 has not been tested in vivo.…”

“…Currently there is a DCLK1 inhibitor, DCLK1-IN-1, which has been reported to inhibit DCLK1 activity with high specificity. 32 Nevertheless, DCLK1-IN-1 has not been tested in vivo. This 34 Furthermore, several kinase inhibitors with antitumor activity, such as XMD8-92 (MAPK7 inhibitor), BI-2536 (PLK1 inhibitor), and TAE-684 (ALK inhibitor), demonstrate nonspecific activity, with a comparable affinity toward DCLK1 as much as their target kinases.…”

The kinase activity of the cancer stem cell marker DCLK1 drives gastric cancer progression by reprogramming the stromal tumor landscape

“…The DCLK1 protein contains a functional serine/threonine kinase domain ( 29 ). In order to investigate whether the kinase activity of DCLK1 is required for the remodeling of the tumor stroma, we treated Balb/C hosts carrying xenograft tumors with a specific, selective and potent DCLK1 kinase inhibitor DCLK1-IN-1 (DCLK1i) ( 30, 31 ). Three weeks later, we observed significantly smaller tumors in the cohort of DCLK1i-treated hosts ( Figure 2 A, B ).…”

“…The current study investigates the function of a single full-length isoform of DCLK1, however, three more splice variants have been described, and while all four isoforms contain the kinase domain, two of them are short, “kinase-only”, isoforms and lack the N-terminal DCX domains which mediate DCLK1’s microtubule-associated function ( 29, 31 ). Moreover, we and others have shown that the DCLK1 kinase domain negatively regulates microtubule polymerization, at least in vitro , and the C-terminal autoinhibitory domain (AID) of DCLK1 competes with ATP for access to the catalytic kinase domain of DCLK1, thus negatively regulating the kinase activity of DCLK1 ( 29, 31, 54 ). Notably, cancer-associated mutations in the AID domain lead to elevated kinase activity and reduced microtubule binding ( 54, 55 ).…”

Structure-Guided Prediction of the Functional Impact of DCLK1 Mutations on Tumorigenesis

“…Several reviews have provided in-depth discussions of the consequence of DCLK1 overexpression to cancer progression, including the functional implications of different DCLK1 protein isoforms generated from the same gene, as well as the effects of therapeutic or genetic inhibition. Although some DCLK1 missense mutations were shown to impact protein function and stability [ 51 , 52 , 53 , 54 ], no specific cancer driver mutation in DCLK1 has been identified or functionally validated. Thus, this review aimed to provide a comprehensive overview of DCLK1 isoforms, the specific functions of the individual DCLK1 domains, and to predict the pro-tumorigenic functions from the most frequent somatic DCLK1 missense mutations in humans.…”