Identification of Kukoamine A, Zeaxanthin, and Clexane as New Furin Inhibitors.

The endogenous protease furin is a key protein in many different diseases, such as cancer and infections. For this reason, a wide range of studies has focused on targeting furin from a therapeutic point of view. Our main objective consisted of identifying new compounds that could enlarge the furin inhibitor arsenal; secondarily, we assayed their adjuvant effect in combination with a known furin inhibitor, CMK, which avoids the SARS-CoV-2 S protein cleavage by means of that inhibition. Virtual screening was carried out to identify potential furin inhibitors. The inhibition of physiological and purified recombinant furin by screening selected compounds, Clexane, and these drugs in combination with CMK was assayed in fluorogenic tests by using a specific furin substrate. The effects of the selected inhibitors from virtual screening on cell viability (293T HEK cell line) were assayed by means of flow cytometry. Through virtual screening, Zeaxanthin and Kukoamine A were selected as the main potential furin inhibitors. In fluorogenic assays, these two compounds and Clexane inhibited both physiological and recombinant furin in a dose-dependent way. In addition, these compounds increased physiological furin inhibition by CMK, showing an adjuvant effect. In conclusion, we identified Kukoamine A, Zeaxanthin, and Clexane as new furin inhibitors. In addition, these drugs were able to increase furin inhibition by CMK, so they could also increase its efficiency when avoiding S protein proteolysis, which is essential for SARS-CoV-2 cell infection.

Gasdermin E-derived caspase-3 inhibitors effectively protect mice from acute hepatic failure.

Programmed cell death (PCD), including apoptosis, apoptotic necrosis, and pyroptosis, is involved in various organ dysfunction syndromes. Recent studies have revealed that a substrate of caspase-3, gasdermin E (GSDME), functions as an effector for pyroptosis; however, few inhibitors have been reported to prevent pyroptosis mediated by GSDME. Here, we developed a class of GSDME-derived inhibitors containing the core structure of DMPD or DMLD. Ac-DMPD-CMK and Ac-DMLD-CMK could directly bind to the catalytic domains of caspase-3 and specifically inhibit caspase-3 activity, exhibiting a lower IC50 than that of Z-DEVD-FMK. Functionally, Ac-DMPD/DMLD-CMK substantially inhibited both GSDME and PARP cleavage by caspase-3, preventing apoptotic and pyroptotic events in hepatocytes and macrophages. Furthermore, in a mouse model of bile duct ligation that mimics intrahepatic cholestasis-related acute hepatic failure, Ac-DMPD/DMLD-CMK significantly alleviated liver injury. Together, this study not only identified two specific inhibitors of caspase-3 for investigating PCD but also, more importantly, shed light on novel lead compounds for treating liver failure and organ dysfunctions caused by PCD.

Thermal shift assay to probe melting of thrombin, fibrinogen, fibrin monomer, and fibrin: Gly-Pro-Arg-Pro induces a fibrin monomer-like state in fibrinogen.

BACKGROUND: Thrombin activates fibrinogen and binds the fibrin E-domain (Kd ~ 2.8 µM) and the splice variant γ'-domain (Kd ~ 0.1 µM). We investigated if the loading of D-Phe-Pro-Arg-chloromethylketone inhibited thrombin (PPACK-thrombin) onto fibrin could enhance fibrin stability. METHODS: A 384-well plate thermal shift assay (TSA) with SYPRO-orange provided melting temperatures (Tm) of thrombin, PPACK-thrombin, fibrinogen, fibrin monomer, and fibrin. RESULTS: Large increases in Tm indicated that calcium led to protein stabilization (0 vs. 2 mM Ca2+) for fibrinogen (54.0 vs. 62.3 °C) and fibrin (62.3 vs. 72.2 °C). Additionally, active site inhibition with PPACK dramatically increased the Tm of thrombin (58.3 vs. 78.3 °C). Treatment of fibrinogen with fibrin polymerization inhibitor GPRP increased fibrinogen stability by ΔTm = 9.3 °C, similar to the ΔTm when fibrinogen was converted to fibrin monomer (ΔTm = 8.8 °C) or to fibrin (ΔTm = 10.4 °C). Addition of PPACK-thrombin at high 5:1 M ratio to fibrin(ogen) had little effect on fibrin(ogen) Tm values, indicating that thrombin binding does not detectably stabilize fibrin via a putative bivalent E-domain to γ'-domain interaction. CONCLUSIONS: TSA was a sensitive assay of protein stability and detected: (1) the effects of calcium-stabilization, (2) thrombin active site labeling, (3) fibrinogen conversion to fibrin, and (4) GPRP induced changes in fibrinogen stability being essentially equivalent to that of fibrin monomer or polymerized fibrin. SIGNIFICANCE: The low volume, high throughput assay has potential for use in understanding interactions with rare or mutant fibrin(ogen) variants.

Formulation and Characterization of Antithrombin Perfluorocarbon Nanoparticles.

Thrombin, a major protein involved in the clotting cascade by the conversion of inactive fibrinogen to fibrin, plays a crucial role in the development of thrombosis. Antithrombin nanoparticles enable site-specific anticoagulation without increasing bleeding risk. Here we outline the process of making and the characterization of bivalirudin and D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone (PPACK) nanoparticles. Additionally, the characterization of these nanoparticles, including particle size, zeta potential, and quantification of PPACK/bivalirudin loading, is also described.

Crystal structures of the recombinant ß-factor XIIa protease with bound Thr-Arg and Pro-Arg substrate mimetics.

Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto-activated forms factor XIIa (FXIIa). Here, the production of the recombinant FXIIa protease domain (ßFXIIaHis) with yields of ∼1-2 mg per litre of insect-cell culture is reported. A second construct utilized an N-terminal maltose-binding protein (MBP) fusion (MBP-ßFXIIaHis). Crystal structures were determined of MBP-ßFXIIaHis in complex with the inhibitor D-Phe-Pro-Arg chloromethyl ketone (PPACK) and of ßFXIIaHis in isolation. The ßFXIIaHis structure revealed that the S2 and S1 pockets were occupied by Thr and Arg residues, respectively, from an adjacent molecule in the crystal. The Thr-Arg sequence mimics the P2-P1 FXIIa cleavage-site residues present in the natural substrates prekallikrein and FXII, and Pro-Arg (from PPACK) mimics the factor XI cleavage site. A comparison of the ßFXIIaHis structure with the available crystal structure of the zymogen-like FXII protease revealed large conformational changes centred around the S1 pocket and an alternate conformation for the 99-loop, Tyr99 and the S2 pocket. Further comparison with activated protease structures of factors IXa and Xa, which also have the Tyr99 residue, reveals that a more open form of the S2 pocket only occurs in the presence of a substrate mimetic. The FXIIa inhibitors EcTI and infestin-4 have Pro-Arg and Phe-Arg P2-P1 sequences, respectively, and the interactions that these inhibitors make with ßFXIIa are also described. These structural studies of ßFXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and anti-inflammatory agents.

Molecular mechanisms of platelet activation and aggregation induced by breast cancer cells.

Tumor cell-induced platelet aggregation represents a critical process both for successful metastatic spread of the tumor and for the development of thrombotic complications in cancer patients. To get further insights into this process, we investigated and compared the molecular mechanisms of platelet aggregation induced by two different breast cancer cell lines (MDA-MB-231 and MCF7) and a colorectal cancer cell line (Caco-2). All the three types of cancer cells were able to induce comparable platelet aggregation, which, however, was observed exclusively in the presence of CaCl2 and autologous plasma. Aggregation was supported both by fibrinogen binding to integrin αIIbß3 as well as by fibrin formation, and was completely prevented by the serine protease inhibitor PPACK. Platelet aggregation was preceded by generation of low amounts of thrombin, possibly through tumor cells-expressed tissue factor, and was supported by platelet activation, as revealed by stimulation of phospholipase C, intracellular Ca2+ increase and activation of Rap1b GTPase. Pharmacological inhibition of phospholipase C, but not of phosphatidylinositol 3-kinase or Src family kinases prevented tumor cell-induced platelet aggregation. Tumor cells also induced dense granule secretion, and the stimulation of the P2Y12 receptor by released ADP was found to be necessary for complete platelet aggregation. By contrast, prevention of thromboxane A2 synthesis by aspirin did not alter the ability of all the cancer cell lines analyzed to induce platelet aggregation. These results indicate that tumor cell-induced platelet aggregation is not related to the type of the cancer cells or to their metastatic potential, and is triggered by platelet activation and secretion driven by the generation of small amount of thrombin from plasma and supported by the positive feedback signaling through secreted ADP.

Quantification of vascular damage in acute kidney injury with fluorine magnetic resonance imaging and spectroscopy.

PURPOSE: To design a fluorine MRI/MR spectroscopy approach to quantify renal vascular damage after ischemia-reperfusion injury, and the therapeutic response to antithrombin nanoparticles (NPs) to protect kidney function. METHODS: A total of 53 rats underwent 45 min of bilateral renal artery occlusion and were treated at reperfusion with either plain perfluorocarbon NPs or NPs functionalized with a direct thrombin inhibitor (PPACK:phenyalanine-proline-arginine-chloromethylketone). Three hours after reperfusion, kidneys underwent ex vivo fluorine MRI/MR spectroscopy at 4.7 T to quantify the extent and volume of trapped NPs, as an index of vascular damage and ischemia-reperfusion injury. Microscopic evaluation of structural damage and NP trapping in non-reperfused renal segments was performed. Serum creatinine was quantified serially over 7 days. RESULTS: The damaged renal cortico-medullary junction trapped a significant volume of NPs (P = 0.04), which correlated linearly (r = 0.64) with the severity of kidney injury 3 h after reperfusion. Despite global large vessel reperfusion, non-reperfusion in medullary peritubular capillaries was confirmed by MRI and microscopy, indicative of continuing hypoxia due to vascular compromise. Treatment of animals with PPACK NPs after acute kidney injury did not accelerate kidney functional recovery. CONCLUSIONS: Quantification of ischemia-reperfusion injury after acute kidney injury with fluorine MRI/MR spectroscopy of perfluorocarbon NPs objectively depicts the extent and severity of vascular injury and its linear relationship to renal dysfunction. The lack of kidney function improvement after early posttreatment thrombin inhibition confirms the rapid onset of ischemia-reperfusion injury as a consequence of vascular damage and non-reperfusion. The prolongation of medullary ischemia renders cortico-medullary tubular structures susceptible to continued necrosis despite restoration of large vessel flow, which suggests limitations to acute interventions after acute kidney injury, designed to interdict renal tubular damage. Magn Reson Med 79:3144-3153, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

An unexpected switch in peptide binding mode: from simulation to substrate specificity.

A ten microsecond molecular dynamics simulation of a kallikrein-related peptidase 7 peptide complex revealed an unexpected change in binding mode. After more than two microseconds unrestrained sampling we observe a spontaneous transition of the binding pose including a 180° rotation around the P1 residue. Subsequently, the substrate peptide occupies the prime side region rather than the cognate non-prime side in a stable conformation. We characterize the unexpected binding mode in terms of contacts, solvent-accessible surface area, molecular interactions and energetic properties. We compare the new pose to inhibitor-bound structures of kallikreins with occupied prime side and find that a similar orientation is adopted. Finally, we apply in silico mutagenesis based on the alternative peptide binding position to explore the prime side specificity of kallikrein-related peptidase 7 and compare it to available experimental data. Our study provides the first microsecond time scale simulation data on a kallikrein protease and shows previously unexplored prime side interactions. Therefore, we expect our study to advance the rational design of inhibitors targeting kallikrein-related peptidase 7, an emerging drug target involved in several skin diseases as well as cancer.

In Vivo-Near Infrared Imaging of Neurodegeneration.

In vivo near-infrared (NIR) imaging of molecular processes at the preclinical stage promises to provide more valuable mechanistic information about pathological pathways involved in neurodegeneration. NIR imaging has the potential to improve in vivo therapeutic screening protocols by enabling noninvasive monitoring of presymptomatic responses to treatment. We have developed new NIR fluorescent contrast agents conjugated to markers of cell death, and using these agents we have identified molecular pathways associated with prion-induced neurodegeneration and determined the optimal window for meaningful therapeutic intervention in prion disease. This chapter provides a description of the synthesis and purification of our NIR cell Death (NIRD) contrast agent and the application of in vivo NIRD (iNIRD) imaging to a prion model of neurodegeneration.

Identification of a degrading enzyme in human serum that hydrolyzes a C-terminal core sequence of neuromedin U.

Neuromedin U (NMU), an anorexigenic peptide, has attracted attention as a candidate for development of drugs against obesity. We recently developed several potent hexapeptidic agonists derived from NMU that share a common Pro-Arg-Asn-NH2 (PRN) sequence at their C-termini and found that the amide bond between Arg and Asn is rapidly degraded in serum. In this study, we determined that the key enzyme responsible for this biodegradation was thrombin. Both irreversible and reversible thrombin inhibitors (PPACK and argatroban, respectively) enhanced the serum stability of both hexapeptidic agonists and human NMU itself as an inherent ligand. In addition, rapid degradation did not occur in citrated human plasma because thrombin was not activated under these conditions. Furthermore, we found that an N-terminal 2-thienylacetyl group in hexapeptidic agonists enhanced recognition by thrombin. These findings will be valuable for future investigations of the biological functions of NMU in vivo. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 440-445, 2016.

Antithrombin nanoparticles inhibit stent thrombosis in ex vivo static and flow models.

OBJECTIVE: Despite significant advances in intravascular stent technology, safe prevention of stent thrombosis over prolonged periods after initial deployment persists as a medical need to decrease device failure. The objective of this project was to assess the potential of perfluorocarbon nanoparticles (NP) conjugated with the direct thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone (PPACK-NP) to inhibit stent thrombosis. METHODS: In a static model of stent thrombosis, 3 × 3-mm pieces of stainless steel coronary stents were cut and adsorbed with thrombin to create a procoagulant surface that would facilitate thrombus development. After treatment with PPACK-NP or control NP, stents were exposed to platelet-poor plasma (PPP) or platelet-rich plasma (PRP) for set time points up to 60 minutes. Measurements of final clot weight in grams were used for assessing the effect of NP treatment on limiting thrombosis. Additionally, groups of stents were exposed to flowing plasma containing various treatments (saline, free PPACK, control NP, and PPACK-NP) and generated thrombi were stained and imaged to investigate the treatment effects of PPACK-NP under flow conditions. RESULTS: The static model of stent thrombosis used in this study indicated a significant reduction in thrombus deposition with PPACK-NP treatment (0.00067 ± 0.00026 g; n = 3) compared with control NP (0.0098 ± 0.0015 g; n = 3; P = .026) in PPP. Exposure to PRP demonstrated similar effects with PPACK-NP treatment (0.00033 ± 0.00012 g; n = 3) vs control NP treatment (0.0045 ± 0.00012 g; n = 3; P = .000017). In additional studies, stents were exposed to both PRP pretreated with vorapaxar and PPACK-NP, which illustrated adjunctive benefit to oral platelet inhibitors for prevention of stent thrombosis. Additionally, an in vitro model of stent thrombosis under flow conditions established that PPACK-NP treatment inhibited thrombus deposition on stents significantly. CONCLUSIONS: This study demonstrates that antithrombin perfluorocarbon NPs exert marked focal antithrombin activity to prevent intravascular stent thrombosis and occlusion.

Thrombomodulin Binding Selects the Catalytically Active Form of Thrombin.

Human α-thrombin is a serine protease with dual functions. Thrombin acts as a procoagulant, cleaving fibrinogen to make the fibrin clot, but when bound to thrombomodulin (TM), it acts as an anticoagulant, cleaving protein C. A minimal TM fragment consisting of the fourth, fifth, and most of the sixth EGF-like domain (TM456m) that has been prepared has much improved solubility, thrombin binding capacity, and anticoagulant activity versus those of previous TM456 constructs. In this work, we compare backbone amide exchange of human α-thrombin in three states: apo, D-Phe-Pro-Arg-chloromethylketone (PPACK)-bound, and TM456m-bound. Beyond causing a decreased level of amide exchange at their binding sites, TM and PPACK both cause a decreased level of amide exchange in other regions including the γ-loop and the adjacent N-terminus of the heavy chain. The decreased level of amide exchange in the N-terminus of the heavy chain is consistent with the historic model of activation of serine proteases, which involves insertion of this region into the ß-barrel promoting the correct conformation of the catalytic residues. Contrary to crystal structures of thrombin, hydrogen-deuterium exchange mass spectrometry results suggest that the conformation of apo-thrombin does not yet have the N-terminus of the heavy chain properly inserted for optimal catalytic activity, and that binding of TM allosterically promotes the catalytically active conformation.

Synthesis and characterization of (18)F-labeled active site inhibited factor VII (ASIS).

Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an (18)F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[(18)F]fluorobenzoate, and the [(18)F]ASIS was purified on a PD-10 desalting column. The radiochemical yield was 25 ± 6%, the radiochemical purity was >97%, and the pseudospecific radioactivity was 35 ± 9 GBq/µmol. The binding efficacy was evaluated in pull-down experiments, which monitored the binding of unlabeled ASIS and [(18)F]ASIS to TF and to a specific anti-factor VII antibody (F1A2-mAb). No significant difference in binding efficacy between [(18)F]ASIS and ASIS could be detected. Furthermore, [(18)F]ASIS was relatively stable in vitro and in vivo in mice. In conclusion, [(18)F]ASIS has for the first time been successfully synthesized as a possible positron emission tomography tracer to image TF expression levels. In vivo positron emission tomography studies to evaluate the full potential of [(18)F]ASIS are in progress.

Ras/ERK signaling pathway is involved in curcumin-induced cell cycle arrest and apoptosis in human gastric carcinoma AGS cells.

Curcumin, the biologically active compound from the rhizome of Curcuma longa, could inhibit cell growth and induce apoptosis in gastric carcinoma. However, the underlying mechanism of curcumin on gastric carcinoma cells still needs further investigation. In this study, morphological observation indicated that curcumin inhibited the proliferation of AGS cells in a dose-dependent manner. According to the flow cytometric analysis, curcumin treatment resulted in G2/M arrest in AGS cells, accompanied with an increased expression of cyclin B1 and a decreased expression of cyclin D1. In addition, DNA ladders were observed by gel electrophoresis. Meanwhile, the activities of caspase-3, -8, and -9 were also enhanced in curcumin-treated AGS cells. Nevertheless, the increased activities could be inhibited by benzyloxycarbonyl-Val-Ala-Asp (OME)-fluoromethylketone (z-VAD-fmk), which suggested that the apoptosis was caspase-dependent. Furthermore, downregulation of rat sarcoma (Ras) and upregulation of extracellular-signal-regulated kinase (ERK) were also observed in AGS cells treated with curcumin by Western blot. U0126, an ERK inhibitor, blocked curcumin-induced apoptosis. The results suggested that curcumin inhibited the growth of the AGS cells and induced apoptosis through the activation of Ras/ERK signaling pathway and downstream caspase cascade, and curcumin might be a potential target for the treatment of gastric carcinoma.

Visualizing cancer and response to therapy in vivo using Cy5.5-labeled factor VIIa and anti-tissue factor antibody.

We have developed a specific technique for imaging cancer in vivo using Cy5.5-labeled factor VIIa (fVIIa), clotting-deficient FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-tissue factor (TF) antibody. FVIIa is the natural ligand for TF. We took advantage of the fact that vascular endothelial cells (VECs) in cancer, but not normal tissue, aberrantly express TF due to its induction by vascular endothelial growth factor (VEGF). Under physiological conditions, TF is expressed by stromal cells and outer blood vessel layers (smooth muscle and adventitia), but not by VECs. We hypothesized that labeled fVIIa or anti-TF antibodies could be used to image the tumor vasculature in vivo. To test this, Cy5.5-labeled fVIIa, FFRck-fVIIa, paclitaxel-FFRck-fVIIa, and anti-TF antibody were developed and administered to athymic nude mice carrying xenografts including glioma U87EGFRviii, pancreatic cancer ASPC-1 and Mia PaCa-2, and squamous cell carcinoma KB-V1. Cy5.5 labeled with these targeting proteins specifically localized to the tumor xenografts for at least 14 days but unconjugated Cy5.5 did not localize to any xenografts or organs. This method of imaging TF in the tumor VECs may be useful in detecting primary tumors and metastases as well as monitoring in vivo therapeutic responses.

Synthetic ion transporters can induce apoptosis by facilitating chloride anion transport into cells.

Anion transporters based on small molecules have received attention as therapeutic agents because of their potential to disrupt cellular ion homeostasis. However, a direct correlation between a change in cellular chloride anion concentration and cytotoxicity has not been established for synthetic ion carriers. Here we show that two pyridine diamide-strapped calix[4]pyrroles induce coupled chloride anion and sodium cation transport in both liposomal models and cells, and promote cell death by increasing intracellular chloride and sodium ion concentrations. Removing either ion from the extracellular media or blocking natural sodium channels with amiloride prevents this effect. Cell experiments show that the ion transporters induce the sodium chloride influx, which leads to an increased concentration of reactive oxygen species, release of cytochrome c from the mitochondria and apoptosis via caspase activation. However, they do not activate the caspase-independent apoptotic pathway associated with the apoptosis-inducing factor. Ion transporters, therefore, represent an attractive approach for regulating cellular processes that are normally controlled tightly by homeostasis.

Single-chain factor XII exhibits activity when complexed to polyphosphate.

BACKGROUND: The mechanism underpinning factor XII autoactivation was originally characterized with non-physiological surfaces, such as dextran sulfate (DS), ellagic acid, and kaolin. Several 'natural' anionic activating surfaces, such as platelet polyphosphate (polyP), have now been identified. OBJECTIVE: To analyze the autoactivation of FXII by polyP of a similar length to that found in platelets (polyP70 ). METHODS AND RESULTS: PolyP70 showed similar efficacy to DS in stimulating autoactivation of FXII, as detected with amidolytic substrate. Western blotting revealed different forms of FXII with the two activating surfaces: two-chain αFXIIa was formed with DS, whereas single-chain FXII (scFXII; 80 kDa) was formed with polyP70 . Dissociation of scFXII from polyP70 abrogated amidolytic activity, suggesting reversible exposure of the active site. Activity of scFXII-polyP70 was enhanced by Zn(2+) and was sensitive to NaCl concentration. A bell-shaped concentration response to polyP70 was evident, as is typical of surface-mediated reactions. Reaction of scFXII-polyP70 with various concentrations of S2302 generated a sigmoidal curve, in contrast to a hyperbolic curve for αFXIIa, from which a Hill coefficient of 3.67 was derived, indicative of positive cooperative binding. scFXII-polyP70 was more sensitive to inhibition by H-d-Pro-Phe-Arg-chloromethylketone and corn trypsin inhibitor than αFXIIa, but inhibition profiles for C1-inhibitor were similar. Active scFXII-polyP70 was also able to cleave its physiological targets FXI and prekallikrein to their active forms. CONCLUSIONS: Autoactivation of FXII by polyP, of the size found in platelets, proceeds via an active single-chain intermediate. scFXII-polyP70 shows activity towards physiological substrates, and may represent the primary event in initiating contact activation in vivo.

A peptide-based positron emission tomography probe for in vivo detection of caspase activity in apoptotic cells.

PURPOSE: Apoptosis, or programmed cell death, can be leveraged as a surrogate measure of response to therapeutic interventions in medicine. Cysteine aspartic acid-specific proteases, or caspases, are essential determinants of apoptosis signaling cascades and represent promising targets for molecular imaging. Here, we report development and in vivo validation of [(18)F]4-fluorobenzylcarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone ([(18)F]FB-VAD-FMK), a novel peptide-based molecular probe suitable for quantification of caspase activity in vivo using positron emission tomography (PET). EXPERIMENTAL DESIGN: Supported by molecular modeling studies and subsequent in vitro assays suggesting probe feasibility, the labeled pan-caspase inhibitory peptide, [(18)F]FB-VAD-FMK, was produced in high radiochemical yield and purity using a simple two-step, radiofluorination. The biodistribution of [(18)F]FB-VAD-FMK in normal tissue and its efficacy to predict response to molecularly targeted therapy in tumors was evaluated using microPET imaging of mouse models of human colorectal cancer. RESULTS: Accumulation of [(18)F]FB-VAD-FMK was found to agree with elevated caspase-3 activity in response to Aurora B kinase inhibition as well as a multidrug regimen that combined an inhibitor of mutant BRAF and a dual PI3K/mTOR inhibitor in (V600E)BRAF colon cancer. In the latter setting, [(18)F]FB-VAD-FMK PET was also elevated in the tumors of cohorts that exhibited reduction in size. CONCLUSIONS: These studies illuminate [(18)F]FB-VAD-FMK as a promising PET imaging probe to detect apoptosis in tumors and as a novel, potentially translatable biomarker for predicting response to personalized medicine.

GpIbα interacts exclusively with exosite II of thrombin.

Activation of platelets by the serine protease thrombin is a critical event in haemostasis. This process involves the binding of thrombin to glycoprotein Ibα (GpIbα) and cleavage of protease-activated receptors (PARs). The N-terminal extracellular domain of GpIbα contains an acidic peptide stretch that has been identified as the main thrombin binding site, and both anion binding exosites of thrombin have been implicated in GpIbα binding, but it remains unclear how they are involved. This issue is of critical importance for the mechanism of platelet activation by thrombin. If both exosites bind to GpIbα, thrombin could potentially act as a platelet adhesion molecule or receptor dimerisation trigger. Alternatively, if only a single site is involved, GpIbα may serve as a cofactor for PAR-1 activation by thrombin. To determine the involvement of thrombin's two exosites in GpIbα binding, we employed the complementary methods of mutational analysis, binding studies, X-ray crystallography and NMR spectroscopy. Our results indicate that the peptide corresponding to the C-terminal portion of GpIbα and the entire extracellular domain bind exclusively to thrombin's exosite II. The interaction of thrombin with GpIbα thus serves to recruit thrombin activity to the platelet surface while leaving exosite I free for PAR-1 recognition.