Lytic Inhibitors Research Articles

Identification of a glycan cluster in gp120 essential for irreversible HIV-1 lytic inactivation by a lectin-based recombinantly engineered protein conjugate.

We previously discovered a class of recombinant lectin conjugates, denoted lectin DLIs ('dual-acting lytic inhibitors') that bind to the HIV-1 envelope (Env) protein trimer and cause both lytic inactivation of HIV-1 virions and cytotoxicity of Env-expressing cells. To facilitate mechanistic investigation of DLI function, we derived the simplified prototype microvirin (MVN)-DLI, containing an MVN domain that binds high-mannose glycans in Env, connected to a DKWASLWNW sequence (denoted 'Trp3') derived from the membrane-associated region of gp41. The relatively much stronger affinity of the lectin component than Trp3 argues that the lectin functions to capture Env to enable Trp3 engagement and consequent Env membrane disruption and virolysis. The relatively simplified engagement pattern of MVN with Env opened up the opportunity, pursued here, to use recombinant glycan knockout gp120 variants to identify the precise Env binding site for MVN that drives DLI engagement and lysis. Using mutagenesis combined with a series of biophysical and virological experiments, we identified a restricted set of residues, N262, N332 and N448, all localized in a cluster on the outer domain of gp120, as the essential epitope for MVN binding. By generating these mutations in the corresponding HIV-1 virus, we established that the engagement of this glycan cluster with the lectin domain of MVN*-DLI is the trigger for DLI-derived virus and cell inactivation. Beyond defining the initial encounter step for lytic inactivation, this study provides a guide to further elucidate DLI mechanism, including the stoichiometry of Env trimer required for function, and downstream DLI optimization.

Label-Free High-Throughput Functional Lytic Assays

Refractive index-sensitive resonant waveguide grating biosensors are used to assay the label-free enzymatic degradation of biomolecules. These assays provide a robust means of screening for functional lytic modulators. The biomolecular substrates in this study were covalently immobilized through amine groups. Using the Corning Epic System, the digestion signatures for multiple protein substrates on the biosensors are measured. Label-free digestion profiles for these proteins were substrate specific. Similarly, the authors find that the label-free digestion is protease specific. Enzyme-substrate pairs were used to evaluate high- throughput biosensors as tools for screening functional modulators. The lytic inhibitor properties for several proteases and dextranase are determined. The authors find that the IC50 values for the protease inhibitors agree with the reported values for several known inhibitors. The Z' values, using biosensor-based functional lytic screens, were routinely greater than 0.5, making this label-free application feasible for high-throughput screening.

Centrifugal pump and heparin coating improves cardiopulmonary bypass biocompatibility

Centrifugal pumps are being used increasingly for short-term extracorporeal circulation purposes such as during heart operations. Whether the centrifugal pump improves the cardiopulmonary bypass biocompatibility has not been fully documented. A roller pump (n = 20) was compared in vivo with a centrifugal pump (n = 20) in groups of patients in which cardiopulmonary bypass circuits that were either totally heparin coated (Carmeda BioActive Surface; n = 20) or uncoated (n = 20) were used. We expected the heparin coating to attenuate blood activation, thus possibly making the comparison of the two pumps easier with respect to their different blood activation potentials. Samples of blood plasma, obtained during cardiopulmonary bypass from low-risk coronary artery bypass grafting patients, were analyzed for hemolysis (plasma haemoglobin), complement activation (C3bc and the terminal complement complex), a complement lytic inhibitor (vitronectin), coagulation activation (fibrinopeptide A), granulocyte activation (lactoferrin), and platelet activation (beta-thromboglobulin). The concentrations of terminal complement complex, lactoferrin, and beta-thromboglobulin were significantly lower in association with heparin-coated surfaces. The concentration of plasma hemoglobin was significantly lower in association with the centrifugal pump. In uncoated circuits, the beta-thromboglobulin level was significantly higher in association with the roller pump than with the centrifugal pump, but this significant reduction in the beta-thromboglobulin level did not hold true for the heparin-coated circuit group. A heparin-coated cardiopulmonary bypass surface reduces the blood activation potential during cardiopulmonary bypass, and the centrifugal pump causes less hemolysis than the roller pump.

Disparity in blood activation by two different heparin-coated cardiopulmonary bypass systems

Several studies have indicated reduced "blood activation" in heparin-coated cardiopulmonary bypass systems. The present study compares the effect of two different heparin coatings on different blood activation indices. Low-risk patients (n = 40) were randomized to coronary artery bypass grafting using cardiopulmonary bypass with surfaces coated entirely by either the Duraflo II heparin coat or the Carmeda Biological Active Surface, or with identical uncoated equipment. In all cases, a standard systemic heparin dosage was used. Complement activation (C3 activation products C3bc and C3a and formation of fluid phase terminal SC5b-9 complement complex), neutrophil activation (lactoferrin and myeloperoxidase), and lytic inhibitors (vitronectin and clusterin) were quantified during cardiopulmonary bypass and 6 hours postoperatively. Heparin coating by either method reduced the formation of terminal SC5b-9 complement complex and the release of lactoferrin and myeloperoxidase compared with uncoated systems. Lactoferrin and myeloperoxidase levels increased significantly during cardiopulmonary bypass in the Duraflo II group, whereas no significant increase was observed in the Carmeda Biological Active Surface group. The least formation of terminal SC5b-9 complement complex and neutrophil activation was observed with the Maxima Carmeda Biological Active Surface-coated equipment. The vitronectin and clusterin concentrations were significantly less reduced in the Duraflo II compared with the control group. This study underlines the importance of terminal SC5b-9 complement complex as a suitable marker in the evaluation of complement activation during cardiopulmonary bypass. Both heparin coatings reduce blood activation, probably more so with Carmeda Biological Active Surface than with Duraflo II.

A novel technique for rapid determination of energy consumption in platelets. Demonstration of different energy consumption associated with three secretory responses.

A novel method has been developed for rapid and quantitative determination of the rate of energy consumption in platelets. In platelets suspended in a cyanide-containing medium. ATP resynthesis is abruptly blocked by addition of 2-deoxyglucose and D-glucono-1,5-lactone. We demonstrate that the subsequent changes in the levels of cytoplasmic ATP and ADP reflect the velocity of energy consumption in the platelets immediately before addition of the inhibitors. Despite the arrest in ATP resynthesis the platelets remain responsive to stimulation by thrombin (5 units x ml-1) which triggers the secretion of the contents of dense, alpha- and acid hydrolase granules. Unstimulated platelets were found to consume about 3.5 and 0.5 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at 37 degrees C and 15 degrees C, respectively; the thrombin-treated platelets consumed respectively 16 and 2 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at these temperatures. When the velocity of energy consumption was varied by (a) changing the temperature and (b) preincubation with glyco(geno)lytic inhibitors, it was found to be linearly related to the initial rate of secretion from the three types of granules. The precise nature of this relationship differed between the three types of secretion responses and indicated an increasing requirement for metabolic energy for secretion from the three types of granules in the order: dense granule less than alpha-granule less than acid hydrolase granule. The results obtained with changes in temperature were superimposable on those obtained with the glyco(geno)lytic inhibitors for dense granule secretion and alpha-granule secretion, suggesting an apparent coupling between energy consumption and the rate of these secretion responses. The rate of secretion of acid hydrolase was always higher when energy consumption was varied by temperature changes than when glyco(geno)lytic inhibitors were used, probably as a result of metabolic changes prior to induction of secretion. On the basis of these experiments, we calculated an incremental energy consumption during complete secretion of dense, alpha- and acid hydrolase granule contents of 2.5, 4.2 and 6.7 mumol of ATP equivalents x (10(11) platelets)-1, respectively.