Binding of Pro-Gly-Pro at the active site of leukotriene A4 hydrolase/aminopeptidase and development of an epoxide hydrolase selective inhibitor

Leukotriene (LT) A₄ hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc metalloenzyme that catalyzes the committed step in the formation of the proinflammatory mediator LTB₄. Recently, the chemotactic tripeptide Pro-Gly-Pro was identified as an endogenous aminopeptidase substrate for LTA₄ hydrolase. Here, we determined the crystal structure of LTA₄ hydrolase in complex with a Pro-Gly-Pro analog at 1.72 Å. From the structure, which includes the catalytic water, and mass spectrometric analysis of enzymatic hydrolysis products of Pro-Gly-Pro, it could be inferred that LTA₄ hydrolase cleaves at the N terminus of the palindromic tripeptide. Furthermore, we designed a small molecule, 4-(4-benzylphenyl)thiazol-2-amine, denoted ARM1, that inhibits LTB₄ synthesis in human neutrophils (IC₅₀ of ∼0.5 μM) and conversion of LTA₄ into LTB₄ by purified LTA4H with a K_i of 2.3 μM. In contrast, 50- to 100-fold higher concentrations of ARM1 did not significantly affect hydrolysis of Pro-Gly-Pro. A 1.62-Å crystal structure of LTA₄ hydrolase in a dual complex with ARM1 and the Pro-Gly-Pro analog revealed that ARM1 binds in the hydrophobic pocket that accommodates the ω-end of LTA₄, distant from the aminopeptidase active site, thus providing a molecular basis for its inhibitory profile. Hence, ARM1 selectively blocks conversion of LTA₄ into LTB₄, although sparing the enzyme’s anti-inflammatory aminopeptidase activity (i.e., degradation and inactivation of Pro-Gly-Pro). ARM1 represents a new class of LTA₄ hydrolase inhibitor that holds promise for improved anti-inflammatory properties.Leukotriene (LT) A₄ hydrolase/aminopeptidase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that catalyzes the formation of the potent chemotactic agent LTB₄, a key lipid mediator in the innate immune response (1, 2). Previous work has shown that LTA₄ hydrolase (LTA4H) is an aminopeptidase with high affinity for N-terminal arginines of various synthetic tripeptides (3, 4). The two enzyme activities of LTA4H are exerted via distinct but overlapping active sites and depend on the catalytic zinc, bound within the signature HEXXH-(X)₁₈-E, typical of M1 metallopeptidases (5–7). In LTA4H, His295, His299, and Glu318 are the zinc-binding ligands, whereas Glu296 is the general base catalyst for peptide hydrolysis (8, 9).LTA4H’s crystal structure has been determined (10). The enzyme folds into an N-terminal domain, a catalytic domain, and a C-terminal domain, each with ∼200 amino acids. The interface of the domains forms a cavity, where the active site is located (Fig. 1). The cavity narrows at the zinc-binding site, forming a tunnel into the catalytic domain. The opening and wider parts of the cavity are highly polar; the tunnel is more hydrophobic. The cavity is mostly defined by the catalytic and C-terminal domains; part of the tunnel is defined by the N-terminal domain. Bound substrate is in contact with all three domains.Open in a separate windowFig. 1.Position and extension of the active center in LTA4H. Cartoon representation of the structure of LTA4H with a tunnel for LTA₄ (red mesh) and peptide substrates (blue mesh). The catalytic zinc (yellow sphere) is located in a wide section of the active site from which a narrow, L-shaped, hydrophobic tunnel protrudes ∼15 Å deeper into the protein. LTA₄ is believed to bind with its ω-end at the end of the hydrophobic tunnel. The volume of the active center was calculated in CAVER (31).Recently, it was discovered that LTA4H cleaves and inactivates the chemotactic tripeptide Pro-Gly-Pro, thus identifying a previously unrecognized endogenous, physiologically significant aminopeptidase substrate (11). Inasmuch as Pro-Gly-Pro attracts neutrophils and promotes inflammation, these data also suggest that LTA4H plays dual and opposite roles during an inflammatory response (i.e., production of chemotactic LTB₄, as well as inactivation of chemotactic Pro-Gly-Pro). Previous efforts to develop inhibitors of LTA4H have used the aminopeptidase activity for screening purposes, and these molecules therefore block both catalytic activities of LTA4H (12).Here, we used crystallography, MS, and a stable peptide analog to determine the binding mode of Pro-Gly-Pro at the active site of LTA4H, as well as the mechanism of peptide cleavage. Based on the structure, we also designed a lead compound that selectively blocks the conversion of LTA₄ into LTB₄, although sparing the hydrolysis of Pro-Gly-Pro.     

Neoatherosclerosis in Patients With Coronary Stent Thrombosis: Findings From Optical Coherence Tomography Imaging (A Report of the PRESTIGE Consortium)

Objectives

The purpose of this study was to assess neoatherosclerosis in a registry of prospectively enrolled patients presenting with stent thrombosis using optical coherence tomography.

A model predicting operative mortality in the UK has only limited value in Denmark

Purpose

Postoperative mortality from colorectal cancer varies between surgical departments. Several models have been developed to predict the operative risk. This study aims to investigate whether the original and the revised Association of Coloproctology of Great Britain and Ireland (ACPGBI) model can predict 30-day mortality after colorectal cancer surgery in Denmark.

Methods

Data were collected from the Danish Colorectal Cancer Group database which has >?95% completeness. All patients operated on from January 2007 to December 2013 were included. The individual estimated operative risk was calculated with the original and revised ACPGBI models. Discrimination and calibration were evaluated with a Receiver Operating Characteristic (ROC) curve analysis and a Hosmer-Lemeshow test, respectively.

Results

In total, 22,807 patients underwent open or laparoscopic colorectal cancer surgery. After excluding 1437 patients because of missing data, 21,370 patients were left for the analyses. The observed 30-day mortality was 5.0%. The original and revised ACPGBI models estimated an operative risk of 7.0 and 4.0%, respectively, with a significant difference in observed and estimated mortality in both models. However, in patients with an estimated risk of at least 26%, i.e., high-risk, good calibration was found with the original ACPGBI model. Discrimination was good with an AUC of 0.83 (95% CI 0.82–0.84) in both models.

Conclusion

The original and revised ACPGBI models are not suitable prediction models for postoperative mortality in the Danish colorectal cancer population. However, the original model might be applicable in predicting mortality in high-risk patients.

     

Allogeneic cell-mediated immunotherapy for eradication of minimal residual disease: comparison of T-cell and IL-2 activated killer (LAK) cell-mediated adoptive immunotherapy in murine models

In the course of allogeneic bone marrow transplantation (BMT), avoiding graft-versus-host disease (GVHD) while retaining the antileukemic effects of the T cells remains a major challenge. T-cell depletion (TCD) reduces the incidence of GVHD but increases the relapse rate after allogeneic BMT. We attempted to develop a regimen that would retain or increase the graft-versus-leukemia effect induced by donor T cells while preventing GVHD. Immunosuppressed mice were given immunocompetent donor cells, i.e., fresh lymphocytes or lymphokine-activated killer (LAK) cells differing from the host in major (MHC) or minor (MiHC) histocompatibility antigens. Engraftment of donor cells was documented by polymerase chain reaction analysis. Administration of MHC- and MiHC-incompatible allogeneic LAK cells, especially in conjunction with recombinant interleukin-2 (rIL-2), increased disease manifestations and mortality associated with GVHD. On the other hand, irradiated LAK cells or TCD-LAK cells prevented GVHD in both mice models studied. Phenotypic analysis of LAK cells demonstrated that CD8(+)-equivalent (Lyt-2) T cells are of significance in aggravation of GVHD. The in vitro cytotoxic capacity of LAK cells against MHC-nonrestricted target cells was not reduced by either irradiation or TCD. These results provide the background for designing improved protocols for immunotherapy of residual disease after BMT. In addition, the data imply that antitumor effects may be retained by irradiated rIL-2-activated allogeneic cells without causing GVHD. Whereas unmodified allogeneic LAK cells can induce more effective graft-versus-leukemia effects at the cost of GVHD, irradiated allogeneic donor LAK cells might play some role in eradication of minimal residual disease following autologous or allogeneic BMT without causing GVHD.     

Inferiority of single-dose sulfadoxine-pyrimethamine intermittent preventive therapy for malaria during pregnancy among HIV-positive Zambian women

BACKGROUND: The World Health Organization advocates 2-3 doses of sulfadoxine-pyrimethamine (SP) for intermittent preventive treatment of malaria (SP IPTp). The optimal number of doses and the consequences of single-dose therapy remain unclear.METHODS: Data were from a randomized, controlled study of human immunodeficiency virus-positive Zambian women comparing monthly versus 2-dose SP IPTp. We compared maternal and neonatal birth outcomes as a function of how many doses the mothers received (1 to > or =4 doses).RESULTS: Of 387 deliveries, 34 received 1 dose of SP. Single-dose SP was significantly associated with higher proportions of maternal anemia, peripheral and cord blood parasitemia, infant prematurity, and low birth weight. SP conferred dose-dependent benefits, particularly in the transition from 1 to 2 doses of SP. Women randomized to the standard 2-dose regimen were much more likely to receive only 1 dose than were women randomized to monthly IPT (relative risk, 16.4 [95% confidence interval, 4.0-68.3]).CONCLUSIONS: Single-dose SP was a common result of trying to implement the standard 2-dose regimen and was inferior to all other dosing regimens. At a programmatic level, this implies that monthly SP IPTp may ultimately be more effective than the standard regimen by reducing the risk of inadvertently underdosing mothers.     

Force-induced polarized mitosis of endothelial and smooth muscle cells in arterial remodeling

Arteries display highly directional growth and remodeling that are specific to increases in the mechanical loads imposed on them by blood pressure, blood flow, and lengthwise tensile forces that are transmitted from the tissues to which they are attached. This study examined the effect of mechanical forces on the direction in which mitosis delivers daughter cells, as a mechanism for directional growth. Lateral forces were imposed on surface integrins of cultured endothelial cells by seeding the cells with arginine-glycine-aspartate peptide-coated magnetic microspheres and applying a magnetic field. Video images revealed that the mitotic axis of dividing cells became highly biased in the direction of applied force. Distribution of cortactin, which participates in polarized mitoses driven by other stimuli, was highly sensitive to mechanical loading and interfering with cortactin function arrested cell growth. Smooth muscle cell mitoses also proved to be sensitive to mechanical force: when lengthwise force imposed on rabbit carotid arteries was altered by excision of a vessel segment and reanastomosis of the cut ends, direction of mitosis was dramatically altered. These findings indicate that influences of mechanical force can modulate the manner in which mitosis of vascular cells contributes to reorganization of arterial wall tissue.     

Two-dose versus monthly intermittent preventive treatment of malaria with sulfadoxine-pyrimethamine in HIV-seropositive pregnant Zambian women

BACKGROUND: Intermittent preventive treatment of malaria during pregnancy (IPTp) reduces placental infection, maternal anemia, and low birth weight (LBW). However, the optimal dosing regimen in settings in which human immunodeficiency virus (HIV) is highly prevalent among pregnant women remains controversial. METHODS: We conducted a randomized, double-blind, placebo-controlled study of IPTp comparing the standard 2-dose sulfadoxine-pyrimethamine (SP) regimen with monthly IPTp among a cohort of HIV-positive pregnant Zambian women. Primary outcomes included placental malaria (by smear and histology) and maternal peripheral parasitemia at delivery. RESULTS: There were no differences between monthly IPTp (n=224) and standard IPTp (n=232) in placental malaria by histopathology (26% vs. 29%; relative risk [RR], 0.90 [95% confidence interval {CI}, 0.64-1.26]) or placental parasitemia (2% vs. 4%; RR, 0.55 [95% CI, 0.17-1.79]). There also were no differences in maternal anemia, stillbirths, preterm delivery, LBW, or all-cause mortality of infants at 6 weeks.CONCLUSIONS: In an area of mesoendemicity in Zambia, monthly SP IPTp was not more efficacious than the standard 2-dose regimen for the prevention of placental malaria or adverse birth outcomes. IPTp policy recommendations need to take into account local malaria transmission patterns and the prevalence of HIV. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00270530.     

Reduction in preterm delivery and neonatal mortality after the introduction of antenatal cotrimoxazole prophylaxis among HIV-infected women with low CD4 cell counts

BACKGROUND: Cotrimoxazole prophylaxis is recommended for subgroups of human immunodeficiency virus (HIV)-infected adults and children to reduce all-cause morbidity and mortality. We investigated whether antenatal cotrimoxazole prophylaxis begun during pregnancy for HIV-infected pregnant women with low CD4 cell counts would affect birth outcomes. METHODS: Cotrimoxazole prophylaxis was introduced as a routine component of antenatal care for HIV-infected women with CD4 cell counts <200 cells/ micro L during the course of a trial of mother-to-child HIV transmission in Lusaka, Zambia. Rates of preterm delivery, low birth weight, and neonatal mortality were compared for women with low CD4 cell counts before and after its introduction. RESULTS: Among 255 women with CD4 cell counts <200 cells/ micro L, the percentage of preterm births (< or =34 weeks of gestation) was lower (odds ratio [OR], 0.49 [95% confidence interval {CI}, 0.24-0.98]) after cotrimoxazole prophylaxis was introduced than before; there was a significant decrease in neonatal mortality (9% to 0%; P=.01) and a trend toward increased birth weight ( beta =114 g [95% CI, -42 to 271 g]). In contrast, there were no significant changes in these parameters over the same time interval among women with CD4 cell counts > or =200 cells/ micro L.Conclusion. Antenatal provision of cotrimoxazole for HIV-infected pregnant women with low CD4 cell counts may have indirect benefits for neonatal health.