Lessons from LIMK1 enzymology and their impact on inhibitor design

LIM domain kinase 1 (LIMK1) is a key regulator of actin dynamics. It is thereby a potential therapeutic target for the prevention of fragile X syndrome and amyotrophic lateral sclerosis. Herein, we use X-ray crystallography and activity assays to describe how LIMK1 accomplishes substrate specificity, to suggest a unique ‘rock-and-poke’ mechanism of catalysis and to explore the regulation of the kinase by activation loop phosphorylation. Based on these findings, a differential scanning fluorimetry assay and a RapidFire mass spectrometry activity assay were established, leading to the discovery and confirmation of a set of small-molecule LIMK1 inhibitors. Interestingly, several of the inhibitors were inactive towards the closely related isoform LIMK2. Finally, crystal structures of the LIMK1 kinase domain in complex with inhibitors (PF-477736 and staurosporine, respectively) are presented, providing insights into LIMK1 plasticity upon inhibitor binding

Platinum(IV)-Loaded Degraded Glycol Chitosan as Efficient Platinum(IV) Drug Delivery Platform

A new class of anticancer prodrugs was designed by combining the cytotoxicity of platinum(IV) complexes and the drug carrier properties of glycol chitosan polymers: Unsymmetrically carboxylated platinum(IV) analogues of cisplatin, carboplatin and oxaliplatin, namely (OC-6-44)-acetatodiammine(3-carboxypropanoato)dichloridoplatinum(IV), (OC-6-44)-acetaodiammine(3-carboxypropanoato)(cyclobutane-1,1-dicarboxylato)platinum(IV) and (OC-6-44)-acetato(3-carboxypropanoato)(1R,2R-cyclohexane-1,2-diamine)oxalatoplatinum(IV) were synthesised and conjugated via amide bonding to degraded glycol chitosan (dGC) polymers with different chain lengths (5, 10, 18 kDa). The 15 conjugates were investigated with 1H and 195Pt NMR spectroscopy, and average amounts of platinum(IV) units per dGC polymer molecule with ICP-MS, revealing a range of 1.3–22.8 platinum(IV) units per dGC molecule. Cytotoxicity was tested with MTT assays in the cancer cell lines A549, CH1/PA-1, SW480 (human) and 4T1 (murine). IC50 values in the low micromolar to nanomolar range were obtained, and higher antiproliferative activity (up to 72 times) was detected with dGC-platinum(IV) conjugates in comparison to platinum(IV) counterparts. The highest cytotoxicity (IC50 of 0.036 ± 0.005 µM) was determined in CH1/PA-1 ovarian teratocarcinoma cells with a cisplatin(IV)–dGC conjugate, which is hence 33 times more potent than the corresponding platinum(IV) complex and twice more potent than cisplatin. Biodistribution studies of an oxaliplatin(IV)–dGC conjugate in non-tumour-bearing Balb/C mice showed an increased accumulation in the lung compared to the unloaded oxaliplatin(IV) analogue, arguing for further activity studies

Quaternary Ammonium Palmitoyl Glycol Chitosan (GCPQ) Loaded with Platinum-Based Anticancer Agents—A Novel Polymer Formulation for Anticancer Therapy

Quaternary ammonium palmitoyl glycol chitosan (GCPQ) has already shown beneficial drug delivery properties and has been studied as a carrier for anticancer agents. Consequently, we synthesised cytotoxic platinum(IV) conjugates of cisplatin, carboplatin and oxaliplatin by coupling via amide bonds to five GCPQ polymers differing in their degree of palmitoylation and quaternisation. The conjugates were characterised by 1H and 195Pt NMR spectroscopy as well as inductively coupled plasma mass spectrometry (ICP-MS), the latter to determine the amount of platinum(IV) units per GCPQ polymer. Cytotoxicity was evaluated by the MTT assay in three human cancer cell lines (A549, non-small-cell lung carcinoma; CH1/PA-1, ovarian teratocarcinoma; SW480, colon adenocarcinoma). All conjugates displayed a high increase in their cytotoxic activity by factors of up to 286 times compared to their corresponding platinum(IV) complexes and mostly outperformed the respective platinum(II) counterparts by factors of up to 20 times, also taking into account the respective loading of platinum(IV) units per GCPQ polymer. Finally, a biodistribution experiment was performed with an oxaliplatin-based GCPQ conjugate in non-tumour-bearing BALB/c mice revealing an increased accumulation in lung tissue. These findings open promising opportunities for further tumouricidal activity studies especially focusing on lung tissue

LIM domain kinase 1 (LIMK1), human kinase domain; A Target Enabling Package

A description of the materials and methods is included within the TEP datasheet. Loss of the translational repressor FMRP in fragile X syndrome causes upregulation of the type II BMP receptor BMPR2 and its non-canonical signalling via the kinase LIMK1. LIMK1 performs inhibitory phosphorylation on cofilin proteins blocking their actin-severing activity. Excessive BMPR2-LIMK1 activation was associated with dendritic spine and behavioural defects in animal models that could be rescued by BMPR2 knockdown or LIMK1 inhibition. Here we present a target enabling package for the therapeutic target LIMK1. We include crystal structures of BMPR2, LIMK1, LIMK2 and the LIMK1-cofilin complex, as well as multiple assays for small molecule inhibitor screening. Finally, we identify a series of allosteric LIMK1 inhibitors with promising potency and selectivity that may potentially allow the development of a safe drug for this chronic indication

High-throughput purification of protein kinases from escherichia coli and insect cells

Protein kinases are major targets for the development of new medicines and play key roles in cellular signaling. The flexible nature of these proteins, posttranslational modifications, and the large size of some protein kinases pose a particular challenge obtaining homogeneous, active recombinant protein kinases suitable for functional or structural studies. Here we describe our expertise expressing protein kinases in two frequently used host systems: E. coli and insect cells using the baculovirus expression vector system. In particular, we will discuss and provide detailed methods on construct design, high-throughput cloning, parallel expression testing and scale up as well as purification and co-expression strategies leading to stable and homogeneous recombinant protein samples

High-throughput purification of protein kinases from escherichia coli and insect cells

Protein kinases are major targets for the development of new medicines and play key roles in cellular signaling. The flexible nature of these proteins, posttranslational modifications, and the large size of some protein kinases pose a particular challenge obtaining homogeneous, active recombinant protein kinases suitable for functional or structural studies. Here we describe our expertise expressing protein kinases in two frequently used host systems: E. coli and insect cells using the baculovirus expression vector system. In particular, we will discuss and provide detailed methods on construct design, high-throughput cloning, parallel expression testing and scale up as well as purification and co-expression strategies leading to stable and homogeneous recombinant protein samples

Conformational plasticity of the ULK3 kinase domain

The human protein kinase ULK3 regulates the timing of membrane abscission, thus being involved in exosome budding and cytokinesis. Herein, we present the first high-resolution structures of the ULK3 kinase domain. Its unique features are explored against the background of other ULK kinases. An inhibitor fingerprint indicates that ULK3 is highly druggable and capable of adopting a wide range of conformations. In accordance with this, we describe a conformational switch between the active and an inactive ULK3 conformation, controlled by the properties of the attached small-molecule binder. Finally, we discuss a potential substrate-recognition mechanism of the full-length ULK3 protein