Identification of Inhibitors for a Virally Encoded Protein Kinase by 2 Different Screening Systems: In Vitro Kinase Assay and In-Cell Activity Assay

The human cytomegalovirus (HCMV) protein kinase pUL97 represents an important determinant for viral replication and thus is a promising target for the treatment of HCMV. The authors screened a compound library of nearly 5000 entities based on known kinase inhibitors in 2 distinct ways. A radioactive in vitro kinase assay was performed with recombinant pUL97, purified from baculovirus-infected insect cells, on myelin basic protein-coated FlashPlates. About 20% of all compounds tested inhibited pUL97 kinase activity by more than 50% at a concentration of 10 μM. These hits belonged to various structural classes. To elucidate their potential to inhibit pUL97 in a cellular context, all compounds of the library were also tested in a cell-based activity assay. For this reason, a HEK293 cell line was established that ectopically expressed pUL97. When these cells were incubated with ganciclovir (GCV), pUL97 phosphorylated GCV to its monophosphate, which subsequently became phosphorylated to cytotoxic metabolites by cellular enzymes. Thereby, pUL97 converted cells into a GCV-sensitive phenotype. Inhibition of the pUL97 kinase activity resulted in protection of the cells against the cytotoxic effects of GCV. In total, 199 compounds of the library were cellular active at nontoxic concentrations, and 93 of them inhibited pUL97 in the in vitro kinase assay. Among these, promising inhibitors of HCMV replication were identified. The 2-fold screening system described here should facilitate the development of pUL97 inhibitors into potent drug candidates

Recombinant GPVI-Fc added to single or dual antiplatelet therapy in vitro prevents plaque-induced platelet thrombus formation

The efficiency of current dual antiplatelet therapy might be further improved by its combination with a glycoprotein (GP) VI-targeting strategy without increasing bleeding. GPVI-Fc, a recombinant dimeric fusion protein binding to plaque collagen and concealing binding sites for platelet GPVI, acts as a lesion-focused antiplatelet drug, and does not increase bleeding in vivo. We investigated, whether GPVI-Fc added in vitro on top of acetylsalicylic acid (ASA), the P2Y(12) antagonist ticagrelor, and the fibrinogen receptor antagonist abciximab alone or in combination would increase inhibition of platelet activation by atherosclerotic plaque. Under static conditions, GPVI-Fc inhibited plaque-induced platelet aggregation by 53%, and increased platelet inhibition by ASA (51%) and ticagrelor (64%) to 66% and 80%, respectively. Under arterial flow, GPVI-Fc inhibited plaque-induced platelet aggregation by 57%, and significantly increased platelet inhibition by ASA (28%) and ticagrelor (47%) to about 81% each. The triple combination of GPVI-Fc, ASA and ticagrelor achieved almost complete inhibition of plaque-induced platelet aggregation (93%). GPVI-Fc alone or in combination with ASA or ticagrelor did not increase closure time measured by the platelet function analyzer (PFA)-200. GPVI-Fc added on top of abciximab, a clinically used anti fibrinogen receptor antibody which blocks platelet aggregation, strongly inhibited total (81%) and stable (89%) platelet adhesion. We conclude that GPVI-Fc added on top of single or dual antiplatelet therapy with ASA and/or a P2Y12 antagonist is likely to improve anti-atherothrombotic protection without increasing bleeding risk. In contrast, the strong inhibition of platelet adhesion by GPVI-Fc in combination with GPIIb/Illa inhibitors could be harmful.</p

The GPVI-Fc Fusion Protein Revacept Improves Cerebral Infarct Volume and Functional Outcome in Stroke

<div><p>Objectives</p><p>We examined the effect of Revacept, an Fc fusion protein which is specifically linked to the extracellular domain of glycoprotein VI (GPVI), on thrombus formation after vessel wall injury and on experimental stroke in mice.</p><p>Background</p><p>Several antiplatelet drugs for the treatment of myocardial infarction or ischemic stroke with potent anti-ischemic effects have been developed, but all incur a significant risk of bleeding.</p><p>Methods</p><p>Platelet adhesion and thrombus formation after endothelial injury was monitored in the carotid artery by intra-vital fluorescence microscopy. The morphological and clinical consequences of stroke were investigated in a mouse model with a one hour-occlusion of the middle cerebral artery.</p><p>Results</p><p>Thrombus formation was significantly decreased after endothelial injury by 1 mg/kg Revacept IV, compared to Fc only. 1 mg/kg Revacept IV applied in mice with ischemic stroke immediately before reperfusion significantly improved functional outcome, cerebral infarct size and edema compared to Fc only. Also treatment with 10 mg/kg rtPA was effective, and functional outcome was similar in both treatment groups. The combination of Revacept with rtPA leads to increased reperfusion compared to treatment with either agent alone. In contrast to rtPA, however, there were no signs of increased intracranial bleeding with Revacept. Both rtPA and Revacept improved survival after stroke compared to placebo treatment. Revacept and vWF bind to collagen and Revacept competitively prevented the binding of vWF to collagen.</p><p>Conclusions</p><p>Revacept reduces arterial thrombus formation, reduces cerebral infarct size and edema after ischemic stroke, improves functional and prognostic outcome without intracranial bleeding. Revacept not only prevents GPVI-mediated, but probably also vWF-mediated platelet adhesion and aggregate formation. Therefore Revacept might be a potent and safe tool to treat ischemic complications of stroke.</p></div

Proteomic characterization of the angiogenesis inhibitor SU6668 reveals multiple impacts on cellular kinase signaling

Knowledge about molecular drug action is critical for the development of protein kinase inhibitors for cancer therapy. Here, we establish a chemical proteomic approach to profile the anticancer drug SU6668, which was originally designed as a selective inhibitor of receptor tyrosine kinases involved in tumor vascularization. By employing immobilized SU6668 for the affinity capture of cellular drug targets in combination with mass spectrometry, we identified previously unknown targets of SU6668 including Aurora kinases and TANK-binding kinase 1. Importantly, a cell cycle block induced by SU6668 could be attributed to inhibition of Aurora kinase activity. Moreover, SU6668 potently suppressed antiviral and inflammatory responses by interfering with TANK-binding kinase 1–mediated signal transmission. These results show the potential of chemical proteomics to provide rationales for the development of potent kinase inhibitors, which combine rather unexpected biological modes of action by simultaneously targeting defined sets of both serine/threonine and tyrosine kinases involved in cancer progression

Experimental stroke model in mice.

<p>Flow in the middle cerebral artery (MCA) was measured after insertion of the catheter/filament and the necessary occlusion of the common carotid artery ( = 100%), during occlusion and 30 minutes of reperfusion, detected by a flexible Laser Doppler flow probe attached to the temporal skull. 1 mg/kg body weight Revacept, or the equimolar amount of Fc only (0.33 mg/kg), or rtPA (10 mg/kg), or a combination of Revacept and rtPA were injected intravenously during reperfusion of the MCA. Mean ± SEM are given, n = 8–10 per group. * represents statistical significance of p<0.05 compared to Fc.</p

Detection of parameters of cellular activation and inflammation in brain tissue by in situ immune-histochemistry in mice.

<p>Top panels show representative brains section of mice with standard hematoxillin eosin staining (HE), and specific IgG extravasation after MCA occlusion and stroke. Brains from Revacept- and saline- treated stroke mice and non-ischemic control brain areas are representatively demonstrated. Bar graphs show immuno-fluorescence signals for immunoglobulin G (IgG), macrophages, transforming growth factor beta (TGF beta) and platelet derived growth factor (PDGF) in mice with stroke after MCA occlusion. We compared the effect of Revacept (1 mg/kg) or Fc only (0.33 mg/kg)-treated mice in the infracted left hemisphere and the right hemisphere in the absence of ischemia. The mean integrals of immuno-fluorescence signals in the respective group are shown as arbitrary units ± SEM, n = 3 per group * indicate significant differences of p<0.05 to saline treated mice.</p

In vivo effects of Revacept on thrombus formation.

<p>Effect of Revacept (1 mg/kg body weight) on thrombus area 30 and 60 minutes after vascular injury induced in the left common carotid artery (A) and effect on transient platelet adhesion (B) of Revacept (1 mg/kg) compared to equimolar amount of Fc only (0.33 mg/kg). The agents were injected intravenously (tail vein) before experimental endothelial lesion. To visualize platelet adhesion and thrombus formation, mice received fluorescence-labelled platelets before injection of agents. Imaging of platelet aggregation was assessed in the carotid artery <i>in vivo</i> by using intravital microscopy (IVM). Mean ± SEM are given, n = 7 per group, * represents statistical significance of p<0.05. (C) Effect of Revacept (0.02–5 mg/kg body weight) on thrombus area after experimental deep vascular injury. The thrombus area was investigated post mortem in macroscopic en face preparations of the carotid area (in µm²). Increasing amounts significantly inhibit thrombus formation. (Mean ± SEM of n = 8–9 animals; * represents statistical significance of p<0.05 compared to PBS buffer).</p

Structure and mode of action of Revacept.

<p>(A) Simplified model of platelet adhesion, aggregation and activation and arterial thrombosis at site of vascular injury. Specific interaction of the extracellular matrix with the platelet receptors alpha₂ beta₁; glycoprotein VI (GPVI) and glycoprotein Ib (GPIb). (B) Revacept binds to collagen exposed at vascular injury. Revacept prevents the first steps of von Willebrand Factor (vWF) and collagen-mediated platelet activation and platelet aggregation. Thus, Revacept seems a useful tool for the treatment or prevention of stroke, in which both vWF and collagen-mediated platelet activation play a central role. For reason of clarity, other pathways that are also relevant to platelet activation in stroke have been omitted.</p

Assessment of stroke volume by post-mortem 2,3,5-TTC staining in brain slices from mice 24 hours after reperfusion following a 1 hour transient MCA occlusion.

<p>(A) representative 2,3,5-TTC stained brain slices. The white scale bar corresponds to 1 cm (upper panel). Mean infarct volumes (lower panel). (B) Mean edema volumes. (C) mean bleeding volumes, as assessed by measuring cyano-methemoglobin content of tissue extracts. Means ± SEM from n = 9 animals per group are given, * denotes significant differences of p<0.05 compared to Fc. At the beginning of reperfusion, mice received either Revacept (1 mg/kg) or an equimolar amount of Fc only (0.33 mg/kg), or rtPA (10 mg/kg), or a combination of Revacept and rtPA, intravenously.</p