Potent inhibitors of CDK5 derived from roscovitine: Synthesis,biological evaluation and molecular modelling

Cyclin dependent kinase 5 (CDK5) is a serine/threonine kinase belonging to the cyclin dependent kinase (CDK) family. CDK5 is involved in numerous neuronal diseases (including Alzheimer’s or Parkinson’s diseases, stroke, traumatic brain injury), pain signaling and cell migration. In the present Letter, we describe syntheses and biological evaluations of new 2,6,9-trisubstituted purines, structurally related to roscovitine, a promising CDK inhibitor currently in clinical trials (CDK1/Cyclin B, IC₅₀ = 350 nM; CDK5/p25, IC₅₀ = 200 nM). These new molecules were synthesized using an original Buchwald–Hartwig catalytic procedure; several compounds (3j, 3k, 3l, 3e, 4k, 6b, 6c) displayed potent kinase inhibitory potencies against CDK5 (IC₅₀ values ranging from 17 to 50 nM) and showed significant cell death inducing activities (IC₅₀ values ranging from 2 to 9 μM on SH-SY5Y). The docking of the inhibitors into the ATP binding domain of the CDK5 catalytic site highlighted the discriminatory effect of a hydrogen bond involving the CDK5 Lys-89. In addition, the calculated final energy balances for complexation measured for several inhibitors is consistent with the ranking of the IC₅₀ values. Lastly, we observed that several compounds exhibit submicromolar activities against DYRK1A (dual specificity, tyrosine phosphorylation regulated kinase 1A), a kinase involved in Down syndrome and Alzheimer’s disease (3g, 3h, 4m; IC₅₀ values ranging from 300 to 400 nM).     

A Clinical Evaluation of Statin Pleiotropy: Statins Selectively and Dose-Dependently Reduce Vascular Inflammation

Statins are thought to reduce vascular inflammation through lipid independent mechanisms. Evaluation of such an effect in atherosclerotic disease is complicated by simultaneous effects on lipid metabolism. Abdominal aortic aneurysms (AAA) are part of the atherosclerotic spectrum of diseases. Unlike atherosclerotic occlusive disease, AAA is not lipid driven, thus allowing direct evaluation of putative anti-inflammatory effects. The anti-inflammatory potency of increasing doses (0, 20 or 40 mg/day) simvastatin or atorvastatin was evaluated in 63 patients that were at least 6 weeks on statin therapy and who underwent open AAA repair. A comprehensive analysis using immunohistochemistry, mRNA and protein analyses was applied on aortic wall samples collected during surgery. The effect of statins on AAA growth was analyzed in a separate prospective study in incorporating 142 patients. Both statins equally effectively and dose-dependently reduced aortic wall expression of NFκB regulated mediators (i.e. IL-6 (P<0.001) and MCP-1 (P<0.001)); shifted macrophage polarization towards a M2 phenotype (P<0.0003); selectively reduced macrophage-related markers such as cathepsin K and S (P<0.009 and 0.0027 respectively), and ALOX5 (P<0.0009), and reduced vascular wall NFκB activity (40 mg/day group, P<0.016). No effect was found on other cell types. Evaluation of the clinical efficacy of statins to reduce AAA progression did not indicate an effect of statins on aneurysm growth (P<0.337). Hence, in the context of AAA the clinical relevance of statins pleiotropy appears minimal.     

Spatial distribution of electrical reconnection after pulmonary vein isolation in patients with recurrent paroxysmal atrial fibrillation

Introduction

Recurrence of atrial fibrillation after pulmonary vein isolation (PVI) occurs frequently and may be associated with electrical reconnection of the pulmonary veins (PV). We investigated spatial distribution of electrical reconnection during re-do procedures in patients with paroxysmal atrial fibrillation who had previous successful acute electrical PVI with either single irrigated tip, antral ablation (s-RF; n = 38) or multi-electrode, duty-cycled ablation (PVAC; n = 48).

Methods and Results

EP navigator, mapping and irrigated tip ablation catheters were used in all re-do procedures. Sites of reconnection were assessed in a 12-segment model. Baseline clinical and demographic characteristics were similar in both groups. The number of PVs reconnected per patient was similar in both groups (2.9 ± 0.9 and 3.2 ± 0.7 (p = 0.193), s?RF and PVAC, respectively), and each PV was equally affected. However, the inferior quadrant of the right lower PV was significantly more vulnerable to reconnection after previous PVAC ablation, whereas the superior quadrant of the right upper PV showed significantly more reconnection in the s?RF group.

Conclusion

The overall number of PVs reconnected was equally high in both groups, and each PV was affected equally. However, there were significant differences in the spatial distribution of electrical reconnection. Better understanding of predilection sites for reconnection might help to improve the long-term success rate of PVI.

     

Relevance of UP elements for three strong Bacillus subtilis phage phi29 promoters

Various Escherichia coli promoters contain, in addition to the classical -35 and -10 hexamers, a third recognition element, named the UP element. Located upstream of the -35 box, UP elements stimulate promoter activity by forming a docking site for the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). Accumulating genetic, biochemical and structural information has provided a detailed picture on the molecular mechanism underlying UP element-dependent promoter stimulation in E.coli. However, far less is known about functional UP elements of Bacillus subtilis promoters. Here we analyse the strong early sigma(A)-RNA polymerase-dependent promoters C2, A2c and A2b of the lytic B.subtilis phage phi29. We demonstrate that the phage promoters contain functional UP elements although their contribution to promoter strength is very different. Moreover, we show that the UP element of the A2b promoter, being critical for its activity, is located further upstream of the -35 box than most E.coli UP elements. The importance of the UP elements for the phage promoters and how they relate to other UP elements are discussed.     

A small, cysteine-rich protein secreted by Fusarium oxysporum during colonization of xylem vessels is required for I-3-mediated resistance in tomato

A 12 kDa cysteine-rich protein is secreted by Fusarium oxysporum f. sp. lycopersici during colonization of tomato xylem vessels. Peptide sequences obtained with mass spectrometry allowed identification of the coding sequence. The gene encodes a 32 kDa protein, designated Six1 for secreted in xylem 1. The central part of Six1 corresponds to the 12 kDa protein found in xylem sap of infected plants. A mutant that had gained virulence on a tomato line with the I-3 resistance gene was found to have lost the SIX1 gene along with neighbouring sequences. Transformation of this mutant with SIX1 restored avirulence on the I-3 line. Conversely, deletion of the SIX1 gene in a wild-type strain results in breaking of I-3-mediated resistance. These results suggest that I-3-mediated resistance is based on recognition of Six1 secreted in xylem vessels.     

Meridianins, a new family of protein kinase inhibitors isolated from the ascidian Aplidium meridianum

Meridianins are brominated 3-(2-aminopyrimidine)-indoles which are purified from Aplidium meridianum, an Ascidian from the South Atlantic (South Georgia Islands). We here show that meridianins inhibit various protein kinases such as cyclin-dependent kinases, glycogen synthase kinase-3, cyclic nucleotide-dependent kinases and casein kinase 1. Meridianins prevent cell proliferation and induce apoptosis, a demonstration of their ability to enter cells and to interfere with the activity of kinases important for cell division and cell death. These results suggest that meridianins constitute a promising scaffold from which more potent and selective protein kinase inhibitors could be designed.     

Dissemination of rat cytomegalovirus through infected granulocytes and monocytes in vitro and in vivo

The role of leukocytes in the in vivo dissemination of cytomegalovirus was studied in this experiment. Rat cytomegalovirus (RCMV) could be transferred to rat granulocytes and monocytes by cocultivation with RCMV-infected fibroblasts in vitro. Intravenous injection of purified infected granulocytes or monocytes resulted in a systemic infection in rats, indicating that our model is a powerful tool to gain further insight into CMV dissemination and the development of new antivirals.     

Pharmacological cyclin-dependent kinase inhibitors inhibit replication of wild-type and drug-resistant strains of herpes simplex virus and human immunodeficiency virus type 1 by targeting cellular,not viral,proteins

Pharmacological cyclin-dependent kinase (cdk) inhibitors (PCIs) block replication of several viruses, including herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus type 1 (HIV-1). Yet, these antiviral effects could result from inhibition of either cellular cdks or viral enzymes. For example, in addition to cellular cdks, PCIs could inhibit any of the herpesvirus-encoded kinases, DNA replication proteins, or proteins involved in nucleotide metabolism. To address this issue, we asked whether purine-derived PCIs (P-PCIs) inhibit HSV and HIV-1 replication by targeting cellular or viral proteins. P-PCIs inhibited replication of HSV-1 and -2 and HIV-1, which require cellular cdks to replicate, but not vaccinia virus or lymphocytic choriomeningitis virus, which are not known to require cdks to replicate. P-PCIs also inhibited strains of HSV-1 and HIV-1 that are resistant to conventional antiviral drugs, which target viral proteins. In addition, the anti-HSV effects of P-PCIs and a conventional antiherpesvirus drug, acyclovir, were additive, demonstrating that the two drugs act by distinct mechanisms. Lastly, the spectrum of proteins that bound to P-PCIs in extracts of mock- and HSV-infected cells was the same. Based on these observations, we conclude that P-PCIs inhibit virus replication by targeting cellular, not viral, proteins.