The prominent neuropathological characteristics of Alzheimer's disease (AD) are neurofibrillary tangles (NFT) and senile plaques.[1-3] The main components of neurofibrillary tangles are abnormally hyperphosphorylated forms of the microtubule-binding protein tau, while brain tau has little or no phosphorylation under normal conditions.[4-11] Thus, it is important to identify the kinase responsible for phosphorylation of tau. Cyclin-dependent kinase 5 (cdk5) phosphorylates microtubule-associated proteins, including tau, Munc18, Map2, and DARPP32.[3, 12-14] Dysregulation of cdk5 is reported to play an important role in the pathogenesis of AD. Although in vivo AD hyperphosphorylation of tau may result from the cooperative action of other kinases, in vitro evidence suggested that cdk5-mediated tau phosphorylation may be the critical step. Cdk5 is activated by binding to one of its two activators, p35 or p39. The active form of kinase is a heterodimer of cdk5 and 25 kDa protein p25, which is a truncated form of p35. Many studies have indicated that inhibition of cdk5/p25 may prevent initiation of tau hyperphosphorylation and in turn should preserve destabilization of microtubules and delay the onset of dementia in AD and other related tauopathies.
In recent years, there has been increasing interest in kinases for finding new drug targets and it is estimated that about 30% of all drug discovery projects in the pharmaceutical industry are currently developing protein kinase inhibitors. So, many biochemical and chromatographic assays have been developed to evaluate the enzyme activity and screen the enzyme inhibitors.[19, 20] For cdk5/p25, the widely used detection techniques are radioactive labeling methods[21, 22] and luminescent kinase methods. However, the above methods are usually accompanied with some disadvantages, for instance high costs, time consuming, and they need of radio-isotope labeling substrates or a secondary reaction coupled to the enzymatic conversion. Therefore, the demand for a label-free, non-radioactive assay scheme is high. Mass-spectrometry-based methods as a sensitive, rapid and accurate tool have been successfully and widely used to screen enzyme inhibitors.[22, 25-27] Up to now, no report has been found in the literature using a mass-spectrometry-based method to quantify the cdk5/p25 activities.
In the present study, a new screening method based on ultrahigh-pressure liquid chromatography (UPLC) and triple quadrupole mass spectrometry (TQMS) was developed. It can accurately quantify the amount of analytes by using a multiple reaction monitoring (MRM) function, which monitors the ratio of mass and charge (m/z) of precursor ions and diagnostic product ions. During this process, the synthetic peptide PKTPKKAKKL (an excellent substrate for cdk5/p25[28, 29]) was applied in the enzymatic reaction. Compared with other existing analytical methods, the UPLC/TQMS method can detect the products or substrates directly and quantitatively, avoiding modification of substrates or secondary enzymatic reactions that are irrelevant to target enzyme reaction.
In this study, the experimental conditions including pH value, temperature, reaction time, and the concentration of reagents such as peptide (PKTPKKAKKL) and adenosine-triphosphate (ATP), were first optimized. Subsequently, the validated method was used to evaluate the inhibitory activity of ginsenosides.
Ginseng has been a well-known herbal medicine in China, Korea, Russia, and Japan for thousands of years. In traditional Chinese medicine, ginseng has been used primarily as a treatment for weakness and fatigue. Nowadays, research studies have demonstrated that ginseng has pharmacological effects on the central nervous system as well as cardiovascular, immune, and endocrine systems. Ginsenosides or ginseng saponins are the major active components of ginseng. Recently, increasing evidence of the beneficial effects of ginsenosides on AD has emerged from both in vivo and in vitro experiments.
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- RESULTS AND DISCUSSION
A new UPLC/TQMS method has been developed. The MS-based assay offers excellent accuracy and reproducibility, and is suited for those enzymes with no chromophore substrate or product. This study presents a detailed kinetic analysis for the two substrates of cdk5/p25 (peptide and ATP). In addition, the UPLC/TQMS-based assay directly detects the native product, which is more accurate than the analysis of quenched samples. The assay is validated by the known inhibitor, roscosvitine, and the IC50 is 0.25 μM, which agreed well with a previously reported value. Aside from the kinetic analysis, the UPLC/TQMS assay can be used to screen the potential inhibitors of cdk5/p25.
Sixteen ginsenosides were identified to be able to inhibit the activity of cdk5/p25. The screening results demonstrated that the effects of PPT group ginsenosides were more potent than those of PPD group ginsenosides. Moreover, there was a possible structure–function relationship that the activity will decrease with increasing number of sugar moieties of PPT group ginsenosides. Consequently, this study opens the way to the study of the structure–function relationship of kinase reactions by the UPLC/TQMS method. This method is also an efficient way to determine the inhibition mode of cdk5/p25 inhibitors. There is therefore no doubt that the information obtained will be useful for the future study of the inhibition mechanism of ginsenosides in Alzheimer's disease.