doripenem In conclusion our work provides the preclinical ev
In conclusion, our work provides the preclinical evidence showing the potent in vivo efficacy of AT7519 on chemoresistant cancer doripenem and its underlying mechanisms of action. Our work supports the biological rationale behind the clinical trials initiated with AT7519, particularly for the treatment of patients who develop chemoresistance and relapse.
Conflicts of interest
Acknowledgement This work was supported by a research grant provided by Jingzhou Central Hospital (Grant No. 201606808).
The cyclin-dependent kinase-like 5 gene is located on chromosome X, and its transcript encodes a serine/threonine protein kinase. Mutations of the gene are known to cause a neurodevelopmental disorder accompanied by intractable epilepsy [,]. The clinical features of mutation were regarded as atypical Rett syndrome for a long time, but have recently been considered a CDKL5 specific disease, CDKL5 deficient disorder . Both missense and nonsense mutations are observed with pathogenic variants . Of these mutations, mostly missense mutation sites are identified within the catalytic domain of CDKL5. Consequently, missense mutations are predicted to change CDKL5 activity and lead to disease onset. Indeed, several missense CDKL5 mutations have been characterized as loss-of-function mutations [, , ]. Thus, detecting enzymatic activity of CDKL5 is important for identifying disease-causative mutations. Further, developing a convenient method for detecting CDKL5 activity will aid molecular diagnosis. Accordingly, in this study, we detected CDKL5 activity by autophosphorylation of CDKL5 in an () expression system using phosphate-binding tag molecule (phos-tag) sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) . This is an efficient method for detecting phosphorylated protein without a non-radioisotope (non-RI). Moreover, it is straightforward and rapid for investigating CDKL5 activity. Wild-type (WT) mouse CDKL5 [mCDKL5(WT)] encoded by an expression plasmid (i.e., pET-mCDKL5[WT]) was constructed as described previously . A kinase activity negative mutant [i.e., mCDKL5(KD)], with Lys-42 and -43 substituted to Arg, and mCDKL5 point mutant expression vectors were constructed by inverse PCR  using pET-mCDKL5(WT) as a template. Each mCDKL5 mutant was expressed in an expression system, as described previously . In brief, each pET-mCDKL5 vector was introduced into BL21(DE3) cells by heat shock transformation, and colonized on Luria–Bertani (LB)+Ampicillin (Amp) plates. A single colony was picked and grown in LB + Amp liquid medium at 37 °C until Abs600 reached 0.6–0.8. Then, 0.1 mM isopropyl-β--thiogalactopyranoside was added. After shaking for 6 h, bacteria were harvested by centrifugation (20,000 × for 10 min), suspended in buffer A [20 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 0.05% Tween-20, and 1 mM phenylmethylsulfonyl fluoride], and sonicated. After sonication, cell supernatant was removed by centrifugation (20,000 × at 4 °C for 10 min), and cell debris washed in 200 μl buffer A. Next, cell debris was re-suspended in 200 μl buffer A, and used as the precipitated fraction. This fraction was analyzed by phos-tag SDS–PAGE or SDS–PAGE, with an equal volume of 2 × SDS sample buffer added to the precipitated fraction. Dephosphorylation of CDKL5 was performed at 30 °C for 1 h in a standard reaction mixture (20 μl) consisting of 40 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2 mM MnCl, 2 mM dithiothreitol, 0.01% Tween-40, 10 μl E precipitated fraction, and 2 μg lambda protein phosphatase-His (λPPase-His) . The reaction was stopped by adding an equal volume of 2 × SDS sample buffer. Mixtures were subjected to phos-tag SDS–PAGE or SDS–PAGE, and analyzed by western blotting. SDS–PAGE was performed according to the method of Laemmli , using slab gels consisting of 10% (w/v) acrylamide separating gels. Phos-tag SDS–PAGE was performed according to our previous method , using 7% (w/v) acrylamide separating gels containing 50 μM acrylamide-pendant phos-tag and 10 μM MnCl. For detection of CDKL5 in precipitated fractions, western blot analysis was performed using His-tag antibody (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and whole protein staining was carried out using a Coomassie brilliant blue (CBB) staining kit (Quick-CBB, FUJIFILM Wako).