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.

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.

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.

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.

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.

Substrate and inhibitor-induced dimerization and cooperativity in caspase-1 but not caspase-3.

Caspases are intracellular cysteine-class proteases with aspartate specificity that is critical for driving processes as diverse as the innate immune response and apoptosis, exemplified by caspase-1 and caspase-3, respectively. Interestingly, caspase-1 cleaves far fewer cellular substrates than caspase-3 and also shows strong positive cooperativity between the two active sites of the homodimer, unlike caspase-3. Biophysical and kinetic studies here present a molecular basis for this difference. Analytical ultracentrifugation experiments show that mature caspase-1 exists predominantly as a monomer under physiological concentrations that undergoes dimerization in the presence of substrate; specifically, substrate binding shifts the KD for dimerization by 20-fold. We have created a hemi-active site-labeled dimer of caspase-1, where one site is blocked with the covalent active site inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. This hemi-labeled enzyme is about 9-fold more active than the apo-dimer of caspase-1. These studies suggest that substrate not only drives dimerization but also, once bound to one site in the dimer, promotes an active conformation in the other monomer. Steady-state kinetic analysis and modeling independently support this model, where binding of one substrate molecule not only increases substrate binding in preformed dimers but also drives the formation of heterodimers. Thus, the cooperativity in caspase-1 is driven both by substrate-induced dimerization as well as substrate-induced activation. Substrate-induced dimerization and activation seen in caspase-1 and not in caspase-3 may reflect their biological roles. Whereas caspase-1 cleaves a dramatically smaller number of cellular substrates that need to be concentrated near inflammasomes, caspase-3 is a constitutively active dimer that cleaves many more substrates located diffusely throughout the cell.

Ligand binding to anion-binding exosites regulates conformational properties of thrombin.

Thrombin participates in coagulation, anticoagulation, and initiation of platelet activation. To fulfill its diverse roles and maintain hemostasis, this serine protease is regulated via the extended active site region and anion-binding exosites (ABEs) I and II. For the current project, amide proton hydrogen-deuterium exchange coupled with MALDI-TOF mass spectrometry was used to characterize ligand binding to individual exosites and to investigate the presence of exosite-active site and exosite-exosite interactions. PAR3(44-56) and PAR1(49-62) were observed to bind to thrombin ABE I and then to exhibit long range effects over to ABE II. By contrast, Hirudin(54-65) focused more on ABE I and did not transmit influences over to ABE II. Although these three ligands were each directed to ABE I, they did not promote the same conformational consequences. D-Phe-Pro-Arg-chloromethyl ketone inhibition at the thrombin active site led to further local and long range consequences to thrombin-ABE I ligand complexes with the autolysis loop often most affected. When Hirudin(54-65) was bound to ABE I, it was still possible to bind GpIbα(269-286) or fibrinogen γ'(410-427) to ABE II. Each ligand exerted its predominant influences on thrombin and also allowed interexosite communication. The results obtained support the proposal that thrombin is a highly dynamic protein. The transmission of ligand-specific local and long range conformational events is proposed to help regulate this multifunctional enzyme.

Structural basis of thrombin-mediated factor V activation: the Glu666-Glu672 sequence is critical for processing at the heavy chain-B domain junction.

Thrombin-catalyzed activation of coagulation factor V (FV) is an essential positive feedback reaction within the blood clotting system. Efficient processing at the N- (Arg(709)-Ser(710)) and C-terminal activation cleavage sites (Arg(1545)-Ser(1546)) requires initial substrate interactions with 2 clusters of positively charged residues on the proteinase surface, exosites I and II. We addressed the mechanism of activation of human factor V (FV) using peptides that cover the entire acidic regions preceding these cleavage sites, FV (657-709)/ (FVa2) and FV(1481-1545)/(FVa3). FVa2 appears to interact mostly with exosite I, while both exosites are involved in interactions with the C-terminal linker. The 1.7-Å crystal structure of irreversibly inhibited thrombin bound to FVa2 unambiguously reveals docking of FV residues Glu(666)-Glu(672) to exosite I. These findings were confirmed in a second, medium-resolution structure of FVa2 bound to the benzamidine-inhibited proteinase. Our results suggest that the acidic A2-B domain linker is involved in major interactions with thrombin during cofactor activation, with its more N-terminal hirudin-like sequence playing a critical role. Modeling experiments indicate that FVa2, and likely also FVa3, wrap around thrombin in productive thrombin·FV complexes that cover a large surface of the activator to engage the active site.

Thrombin-targeted liposomes establish a sustained localized anticlotting barrier against acute thrombosis.

The goal of the present work was to design and test an acute-use nanoparticle-based antithrombotic agent that exhibits sustained local inhibition of thrombin without requiring a systemic anticoagulant effect to function against acute arterial thrombosis. To demonstrate proof of concept, we functionalized the surface of liposomes with multiple copies of the direct thrombin inhibitor, d-phenylalanyl-l-prolyl-l-arginyl-chloromethyl ketone (PPACK), which exhibits high affinity for thrombin as a free agent but manifests too rapid clearance in vivo to be effective alone. The PPACK-liposomes were formulated as single unilamellar vesicles, with a diameter of 170.78 ± 10.59 nm and a near neutral charge. In vitro models confirmed the inhibitory activity of PPACK-liposomes, demonstrating a KI' of 172.6 nM. In experimental clots in vitro, treatment of formed clots completely abrogated any further clotting upon exposure to human plasma. The liposomes were evaluated in vivo in a model of photochemical-induced carotid artery injury, resulting in significantly prolonged arterial occlusion time over that of controls (69.06 ± 5.65 min for saline treatment, N = 6, 71.33 ± 9.46 min for free PPACK treated; N = 4, 85.75 ± 18.24 min for precursor liposomes; N = 4, 139.75 ± 20.46 min for PPACK-liposomes; P = 0.0049, N = 6). Systemic anticoagulant profiles revealed a rapid return to control levels within 50 min, while still maintaining antithrombin activity at the injury site. The establishment of a potent and long-acting anticoagulant surface over a newly forming clot with the use of thrombin targeted nanoparticles that do not require systemic anticoagulation to be effective offers an alternative site-targeted approach to the management of acute thrombosis.

The co-crystal structure of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) with a tripeptide fluoromethyl ketone (Z-VAE(OMe)-FMK).

UCHL1 is a 223 amino acid member of the UCH family of deubiquitinating enzymes (DUBs), found abundantly and exclusively expressed in neurons and the testis in normal tissues. Two naturally occurring variants of UCHL1 are directly involved in Parkinson's disease (PD). Not only has UCHL1 been linked to PD, but it has oncogenic properties, having been found abnormally expressed in lung, pancreatic, and colorectal cancers. Although inhibitors of UCHL1 have been described previously the co-crystal structure of the enzyme bound to any inhibitor has not been reported. Herein, we report the X-ray structure of UCHL1 co-crystallized with a peptide-based fluoromethylketone inhibitor, Z-VAE(OMe)-FMK (VAEFMK) at 2.35 Å resolution. The co-crystal structure reveals that the inhibitor binds in the active-site cleft, irreversibly modifying the active-site cysteine; however, the catalytic histidine is still misaligned as seen in the native structure, suggesting that the inhibitor binds to an inactive form of the enzyme. Our structure also reveals that the inhibitor approaches the active-site cleft from the opposite side of the crossover loop as compared to the direction of approach of ubiquitin's C-terminal tail, thereby occupying the P1' (leaving group) site, a binding site perhaps used by the unknown C-terminal extension of ubiquitin in the actual in vivo substrate(s) of UCHL1. This structure provides a view of molecular contacts at the active-site cleft between the inhibitor and the enzyme as well as furnishing structural information needed to facilitate further design of inhibitors targeted to UCHL1 with high selectivity and potency.

Structure of human caspase-6 in complex with Z-VAD-FMK: New peptide binding mode observed for the non-canonical caspase conformation.

Caspase-6 is a cysteine protease implicated in neuronal survival and apoptosis. Deregulation of caspase-6 activity was linked to several neurodegenerative disorders including Alzheimer's and Huntington's Diseases. Several recent studies on the structure of caspase-6 feature the caspase-6 zymogen, mature apo-caspase-6 as well as the Ac-VEID-CHO peptide complex. All structures share the same typical dimeric caspase conformation. However, mature apo-caspase-6 crystallized at low pH revealed a novel, non-canonical inactive caspase conformation speculated to represent a latent state of the enzyme suitable for the design of allosteric inhibitors. In this treatise we present the structure of caspase-6 in the non-canonical inactive enzyme conformation bound to the irreversible inhibitor Z-VAD-FMK. The complex features a unique peptide binding mode not observed previously.

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.

Synthesis and biological evaluation of modified pentapeptides as potent proteinase K inhibitors.

This communication reports the first demonstration of synthesis and biological validation of modified pentapeptides, such as methoxysuccinyl-Ala-Ala-Ala-Pro-Leu-chloromethyl ketone 6b as a potent proteinase K inhibitor. The efficacy of MeOSuc-AAAPL-CH(2)Cl 6b analog in inhibiting the proteolytic activity of proteinase K was compared with the known MeOSuc-AAPV-CH(2)Cl analog. The examination of inhibitory activity using RT-PCR assay in the presence of proteinase K revealed that the MeOSuc-AAAPL-CH(2)Cl 6b inhibitor at a concentration of 0.05 mM allows a signal to be obtained for an exogenous target ('Xeno RNA') at 30 cycles (i.e., Ct=30), whereas the control MeOSuc-AAPV-CH(2)Cl requires a fivefold higher concentration (0.25 mM) to produce the same Ct. A plausible explanation for the higher efficiency of MeOSuc-AAAPL-CH(2)Cl 6b over control is proposed based on the molecular modeling studies.

Design, synthesis and inhibitory effect of pentapeptidyl chloromethyl ketones on proteinase K.

The synthesis and proteolytic inhibitor function of new modified pentapeptide MeOSuc-AAAPF-CH(2)Cl 6 is described. The efficacy of 6 in inhibiting the proteolytic activity of proteinase K at a concentration of 0.10 mM allows a signal to be obtained for an exogenous target ('Xeno RNA') at 29 PCR cycles (i.e., Ct=29), whereas the control MeOSuc-AAAPV-CH2Cl 1 requires a 7.5-fold higher concentration (0.75 mM) to produce the same Ct.

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.

Proton bridging in the interactions of thrombin with small inhibitors.

Thrombin is the pivotal serine protease enzyme in the blood cascade system. Phe-Pro-Arg-chloromethylketone (PPACK), phosphate, and phosphonate ester inhibitors form a covalent bond with the active-site Ser of thrombin. PPACK, a mechanism-based inhibitor, and the phosphate/phosphonate esters form adducts that mimic intermediates formed in reactions catalyzed by thrombin. Therefore, the dependence of the inhibition of human alpha-thrombin on the concentration of these inhibitors, pH, and temperature was investigated. The second-order rate constant (ki/Ki) and the inhibition constant (Ki) for inhibition of human alpha-thrombin by PPACK are (1.1 +/- 0.2) x 10(7) M(-1) s(-1) and (2.4 +/- 1.3) x 10(-8) M, respectively, at pH 7.00 in 0.05 M phosphate buffer and 0.15 M NaCl at 25.0 +/- 0.1 degrees C, in good agreement with previous reports. The activation parameters at pH 7.00 in 0.05 M phosphate buffer and 0.15 M NaCl are as follows: DeltaH = 10.6 +/- 0.7 kcal/mol, and DeltaS = 9 +/- 2 cal mol(-1) degrees C(-1). The pH dependence of the second-order rate constants of inhibition is bell-shaped. Values of pKa1 and pKa2 are 7.3 +/- 0.2 and 8.8 +/- 0.3, respectively, at 25.0 +/- 0.1 degrees C. A phosphate and a phosphonate ester inhibitor gave higher values, 7.8 and 8.0 for pKa1 and 9.3 and 8.6 for pKa2, respectively. They inhibit thrombin more than 6 orders of magnitude less efficiently than PPACK does. The deuterium solvent isotope effect for the second-order rate constant at pH 7.0 and 8.3 at 25.0 +/- 0.1 degrees C is unity within experimental error in all three cases, indicating the absence of proton transfer in the rate-determining step for the association of thrombin with the inhibitors, but in a 600 MHz 1H NMR spectrum of the inhibition adduct at pH 6.7 and 30 degrees C, a peak at 18.10 ppm with respect to TSP appears with PPACK, which is absent in the 1H NMR spectrum of a solution of the enzyme between pH 5.3 and 8.5. The peak at low field is an indication of the presence of a short-strong hydrogen bond (SSHB) at the active site in the adduct. The deuterium isotope effect on this hydrogen bridge is 2.2 +/- 0.2 (phi = 0.45). The presence of an SSHB is also established with a signal at 17.34 ppm for a dealkylated phosphate adduct of thrombin.

Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death.

In the accompanying paper by Weil et al. (1996) we show that staurosporine (STS), in the presence of cycloheximide (CHX) to inhibit protein synthesis, induces apoptotic cell death in a large variety of nucleated mammalian cell types, suggesting that all nucleated mammalian cells constitutively express all of the proteins required to undergo programmed cell death (PCD). The reliability of that conclusion depends on the evidence that STS-induced, and (STS + CHS)-induced, cell deaths are bona fide examples of PCD. There is rapidly accumulating evidence that some members of the Ced-3/Interleukin-1 beta converting enzyme (ICE) family of cysteine proteases are part of the basic machinery of PCD. Here we show that Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a cell-permeable, irreversible, tripeptide inhibitor of some of these proteases, suppresses STS-induced and (STS + CHX)-induced cell death in a wide variety of mammalian cell types, including anucleate cytoplasts, providing strong evidence that these are all bona fide examples of PCD. We show that the Ced-3/ICE family member CPP32 becomes activated in STS-induced PCD, and that Bcl-2 inhibits this activation. Most important, we show that, in some cells at least, one or more CPP32-family members, but not ICE itself, is required for STS-induced PCD. Finally, we show that zVAD-fmk suppresses PCD in the interdigital webs in developing mouse paws and blocks the removal of web tissue during digit development, suggesting that this inhibition will be a useful tool for investigating the roles of PCD in various developmental processes.