CL-11 circulates in serum as functionally distinct isoforms.

Collectin-11 (CL-11) is a pattern recognition molecule of the lectin pathway capable of interacting with collectin-10 (CL-10) and the MASPs to activate the complement cascade. Alternative splicing of the COLEC11 gene gives rise to two different isoforms found in serum (A and D). These isoforms vary in the length of their collagen-like region, which is involved in the stabilization of the trimeric subunit and the interaction with the MASPs. Here we aim at elucidating the biological differences of naturally occurring CL-11 isoforms A and D. We produced recombinant CL-11 as independent isoforms (CL-11A and CL-11D) and together with CL-10 (CL-10/11A, CL-10/11D). Both CL-11 isoforms associated with CL-10, but CL-11D did so to a lesser extent. CL-10/11 heterocomplexes were composed of trimeric subunits of CL-10 and CL-11, as opposed to CL-10 and CL-11 homotrimers. Heterocomplexes were more stable and migrated with higher apparent molecular weights. Immunoprecipitation of serum CL-11 and subsequent mass spectrometry analysis confirmed that native CL-11 circulates in the form of CL-10/11 heterocomplexes that associate with MASP-1, and MASP-3, but not necessarily MASP-2. Despite a shorter collagen region, CL-11D was capable to bind to the MASPs, suggesting that the missing exon 4 is not required for MASP association CL-11D had a reduced ligand binding compared to full-length CL-11A. Based on its reduced ability to oligomerize, form CL-10/11 heterocomplexes, and bind to ligands, we hypothesize that CL-11D may have a limited complement activation potential compared to full-length CL-11A.

Effects of Hormone Replacement Therapy on Women's Lung Health and Disease.

This review provides an overview of menopausal hormone therapy and pulmonary disease risk, with a focus on the effect of hormone replacement therapy (HRT) on pulmonary function and its relation to lung diseases. This summary is based on authors' knowledge in the field of HRT and supplemented by a PubMed search using the terms "menopause hormone therapy," "asthma", "lung cancer", "chronic obstructive pulmonary disease", "lung function", and "pulmonary hypertension". Available evidence indicates that there is limited research on the role of sex hormones in the susceptibility, severity, and progression of chronic respiratory diseases. However, some studies suggest that the hormonal changes that occur during the menopausal transition may have an impact on pulmonary function and respiratory diseases. Women are in need of convenient access to a safe and effective modality for personalized HRT based on an artificial intelligence (AI)-driven platform that will enable them to receive personalized hormonal treatment through frequent, convenient, and accurate measurements of hormone levels in peripheral blood.

Chronic thoracic pain after cardiac surgery: role of inflammation and biomechanical sternal stability.

Introduction: The pathogenesis of chronic chest pain after cardiac surgery has not been determinate. If left untreated, postoperative sternal pain reduces the quality of life and patient satisfaction with cardiac surgery. The purpose of the study was to examine the effect of chest inflammation on postoperative pain, risk factors for chronic pain after cardiac surgery and to explore how chest reconstruction was associated with the intensity of pain. Methods: The authors performed a study of acute and chronic thoracic pain after cardiac surgery in patients with and without sternal infection and compared different techniques for chest reconstruction. 42 high-risk patients for the development of mediastinitis were included. Patients with mediastinitis received chest reconstruction (group 1). Their demographics and risk factors were matched with no-infection patients with chest reconstruction (group 2) and subjects who underwent conventional sternal closure (group 3). Chronic pain was assessed by the numeric rating scale after surgery. Results: The assessment of the incidence and intensity of chest pain at 3 months post-surgery demonstrated that 14 out of 42 patients across all groups still experienced chronic pain. Specifically, in group 1 with sternal infection five patients had mild pain, while one patient experienced mild pain in group 2, and eight patients in group 3. Also, follow-up results indicated that the highest pain score was in group 3. While baseline levels of cytokines were increased among patients with sternal infection, at discharge only the level of interleukin 6 remained high compared to no infection groups. Compared to conventional closure, after chest reconstruction, we found better healing scores at 3-month follow-up and a higher percentage of patients with the complete sternal union. Conclusions: Overall, 14 out of 42 patients have chronic pain after cardiac surgery. The intensity of the pain in mediastinitis patients significantly decreased at 3 months follow-up after chest reconstruction. Thus, post-surgery mediastinitis is not a determining factor for development the chronic chest pain. There is no correlation between cytokines levels and pain score except interleukin 6 which remains elevated for a long time after treatment. Correlation between sternal healing score and chronic chest pain was demonstrated.

Acid ceramidase gene therapy ameliorates pulmonary arterial hypertension with right heart dysfunction.

BACKGROUND: Up-regulation of ceramides in pulmonary hypertension (PH), contributing to perturbations in sphingolipid homeostasis and the transition of cells to a senescence state. We assessed the safety, feasibility, and efficiency of acid ceramidase gene transfer in a rodent PH model. METHODS: A model of PH was established by the combination of left pneumonectomy and injection of Sugen toxin. Magnetic resonance imaging and right heart catheterization confirmed development of PH. Animals were subjected to intratracheal administration of synthetic adeno-associated viral vector (Anc80L65) carrying the acid ceramidase (Anc80L65.AC), an empty capsid vector, or saline. Therapeutic efficacy was evaluated 8 weeks after gene delivery. RESULTS: Hemodynamic assessment 4 weeks after PH model the development demonstrated an increase in the mean pulmonary artery pressure to 30.4 ± 2.13 mmHg versus 10.4 ± 1.65 mmHg in sham (p < 0.001), which was consistent with the definition of PH. We documented a significant increase in pulmonary vascular resistance in the saline-treated (6.79 ± 0.85 mm Hg) and empty capsid (6.94 ± 0.47 mm Hg) groups, but not in animals receiving Anc80L65.AC (4.44 ± 0.71 mm Hg, p < 0.001). Morphometric analysis demonstrated an increase in medial wall thickness in control groups in comparison to those treated with acid ceramidase. After acid ceramidase gene delivery, a significant decrease of pro-inflammatory factors, interleukins, and senescence markers was observed. CONCLUSION: Gene delivery of acid ceramidase provided tropism to pulmonary tissue and ameliorated vascular remodeling with right ventricular dysfunction in pulmonary hypertension.

Cardiac Targeted Adeno-Associated Virus Injection in Rats.

Gene therapy is a promising approach in the treatment of cardiovascular diseases. The vectors available for cardiovascular gene therapy have significantly improved over time. Cardiac tropism is a primary characteristic of an ideal vector along with a long-term expression profile and a minimal risk of cellular immune response. Preclinical and clinical studies have demonstrated that adeno-associated viral (AAV) vectors are one of the most attractive vehicles for gene transfer. AAV has gained great popularity in the last years because of its biological properties and advantages over other viral vector systems. In this chapter we will describe methods for intracardiac delivery of AAV vector in rats.

Surgical Methods for Cardiac Gene Delivery in Large Animals.

This chapter describes main strategies of surgical gene delivery in large animals. Existing methods of cardiac gene transfer can be classified by the site of injection, interventional approach, and type of cardiac circulation at the time of transfer. Randomized clinical trials have suggested that the therapeutic benefits of gene therapy are not as substantial as expected from animal studies. This discordance in results is largely due to gene delivery methods that may be effective in small animals but are not scalable to larger species and, therefore, cannot transduce a sufficient fraction of myocytes to establish long-term clinical efficacy. Ideally, an optimized gene transfer should incorporate the following: a closed-loop recirculation for extended transgene residence time; vector washout form the vascular system after transfer to prevent collateral expression; use of methods to increase myocardial transcapillary gradient for viral particles for a better transduction, probably retrograde route of gene delivery through the coronary venous system; and myocardial ischemic preconditioning.

HIPK2 directs cell type-specific regulation of STAT3 transcriptional activity in Th17 cell differentiation.

SignificanceSTAT3 (signal transducer and activator of transcription 3) is a master transcription factor that organizes cellular responses to cytokines and growth factors and is implicated in inflammatory disorders. STAT3 is a well-recognized therapeutic target for human cancer and inflammatory disorders, but how its function is regulated in a cell type-specific manner has been a major outstanding question. We discovered that Stat3 imposes self-directed regulation through controlling transcription of its own regulator homeodomain-interacting protein kinase 2 (Hipk2) in a T helper 17 (Th17) cell-specific manner. Our validation of the functional importance of the Stat3-Hipk2 axis in Th17 cell development in the pathogenesis of T cell-induced colitis in mice suggests an approach to therapeutically treat inflammatory bowel diseases that currently lack a safe and effective therapy.

Efficient cardiac gene transfer and early-onset expression of a synthetic adeno-associated viral vector, Anc80L65, after intramyocardial administration.

OBJECTIVE: Gene therapy is a promising approach in the treatment of cardiovascular diseases. Preclinical and clinical studies have demonstrated that adeno-associated viral vectors are the most attractive vehicles for gene transfer. However, preexisting immunity, delayed gene expression, and postinfection immune response limit the success of this technology. The aim of this study was to investigate the efficacy of the first synthetic adeno-associated viral lineage clone, Anc80L65, for cardiac gene therapy. METHODS: By combining 2 different reporter approaches by fluorescence with green fluorescent protein and bioluminescence (Firefly luciferase), we compared transduction efficiency of Anc80L65 and adeno-associated virus, serotype 9 in neonatal rat cardiomyocytes ex vivo and rat hearts in vivo after intramyocardial and intracoronary administration. RESULTS: In cardiomyocytes, Anc80L65 provided a green fluorescent protein expression of 28.9% (36.4 ± 3.34 cells/field) at 24 hours and approximately 100% on day 7. In contrast, adeno-associated virus, serotype 9 green fluorescent protein provided minimal green fluorescent protein expression of 5.64% at 24 hours and 11.8% on day 7. After intramyocardial injection, vector expression peaked on day 7 with Anc80L65; however, with adeno-associated virus, serotype 9 the peak expression was during week 6. Administration of Anc80L65 demonstrated significantly more efficient expression of reporter gene than after adeno-associated virus, serotype 9 at 6 weeks (6.81 ± 0.64 log10 gc/100 ng DNA vs 6.49 ± 0.28 log10 gc/100 ng DNA, P < .05). These results were consistent with the amount of genome copy per cell observed in the heart. CONCLUSIONS: Anc80L65 vector allows fast and robust gene transduction compared with adeno-associated virus, serotype 9 vector in cardiac gene therapy. Anc80L65 did not adversely affect cardiac function and caused no inflammatory response or toxicity.

Bromodomain protein BRD4 directs and sustains CD8 T cell differentiation during infection.

In response to infection, pathogen-specific CD8 T cells differentiate into functionally diverse effector and memory T cell populations critical for resolving disease and providing durable immunity. Through small-molecule inhibition, RNAi studies, and induced genetic deletion, we reveal an essential role for the chromatin modifier and BET family member BRD4 in supporting the differentiation and maintenance of terminally fated effector CD8 T cells during infection. BRD4 bound diverse regulatory regions critical to effector T cell differentiation and controlled transcriptional activity of terminal effector-specific super-enhancers in vivo. Consequentially, induced deletion of Brd4 or small molecule-mediated BET inhibition impaired maintenance of a terminal effector T cell phenotype. BRD4 was also required for terminal differentiation of CD8 T cells in the tumor microenvironment in murine models, which we show has implications for immunotherapies. Taken together, these data reveal an unappreciated requirement for BRD4 in coordinating activity of cis regulatory elements to control CD8 T cell fate and lineage stability.

Altering Sphingolipid Metabolism Attenuates Cell Death and Inflammatory Response After Myocardial Infarction.

BACKGROUND: Sphingolipids have recently emerged as a biomarker of recurrence and mortality after myocardial infarction (MI). The increased ceramide levels in mammalian heart tissues during acute MI, as demonstrated by several groups, is associated with higher cell death rates in the left ventricle and deteriorated cardiac function. Ceramidase, the only enzyme known to hydrolyze proapoptotic ceramide, generates sphingosine, which is then phosphorylated by sphingosine kinase to produce the prosurvival molecule sphingosine-1-phosphate. We hypothesized that Acid Ceramidase (AC) overexpression would counteract the negative effects of elevated ceramide and promote cell survival, thereby providing cardioprotection after MI. METHODS: We performed transcriptomic, sphingolipid, and protein analyses to evaluate sphingolipid metabolism and signaling post-MI. We investigated the effect of altering ceramide metabolism through a loss (chemical inhibitors) or gain (modified mRNA [modRNA]) of AC function post hypoxia or MI. RESULTS: We found that several genes involved in de novo ceramide synthesis were upregulated and that ceramide (C16, C20, C20:1, and C24) levels had significantly increased 24 hours after MI. AC inhibition after hypoxia or MI resulted in reduced AC activity and increased cell death. By contrast, enhancing AC activity via AC modRNA treatment increased cell survival after hypoxia or MI. AC modRNA-treated mice had significantly better heart function, longer survival, and smaller scar size than control mice 28 days post-MI. We attributed the improvement in heart function post-MI after AC modRNA delivery to decreased ceramide levels, lower cell death rates, and changes in the composition of the immune cell population in the left ventricle manifested by lowered abundance of proinflammatory detrimental neutrophils. CONCLUSIONS: Our findings suggest that transiently altering sphingolipid metabolism through AC overexpression is sufficient and necessary to induce cardioprotection post-MI, thereby highlighting the therapeutic potential of AC modRNA in ischemic heart disease.

Inhibitor of CBP Histone Acetyltransferase Downregulates p53 Activation and Facilitates Methylation at Lysine 27 on Histone H3.

Tumor suppressor p53-directed apoptosis triggers loss of normal cells, which contributes to the side-effects from anticancer therapies. Thus, small molecules with potential to downregulate the activation of p53 could minimize pathology emerging from anticancer therapies. Acetylation of p53 by the histone acetyltransferase (HAT) domain is the hallmark of coactivator CREB-binding protein (CBP) epigenetic function. During genotoxic stress, CBP HAT-mediated acetylation is essential for the activation of p53 to transcriptionally govern target genes, which control cellular responses. Here, we present a small molecule, NiCur, which blocks CBP HAT activity and downregulates p53 activation upon genotoxic stress. Computational modeling reveals that NiCur docks into the active site of CBP HAT. On CDKN1A promoter, the recruitment of p53 as well as RNA Polymerase II and levels of acetylation on histone H3 were diminished by NiCur. Specifically, NiCur reduces the levels of acetylation at lysine 27 on histone H3, which concomitantly increases the levels of trimethylation at lysine 27. Finally, NiCur attenuates p53-directed apoptosis by inhibiting the Caspase 3 activity and cleavage of Poly (ADP-ribose) polymerase (PARP) in normal gastrointestinal epithelial cells. Collectively, NiCur demonstrates the potential to reprogram the chromatin landscape and modulate biological outcomes of CBP-mediated acetylation under normal and disease conditions.

Cell type-specific pharmacological kinase inhibition for cancer chemoprevention.

Safety is prerequisite for preventive medicine, but non-toxic agents are generally ineffective as clinical chemoprevention. Here we propose a strategy overcoming this challenge by delivering molecular-targeted agent specifically to the effector cell type to achieve sufficient potency, while circumventing toxicity in the context of cancer chemoprevention. Hepatic myofibroblasts drive progressive fibrosis that results in cirrhosis and liver cancer. In a rat model of cirrhosis-driven liver cancer, a small molecule epidermal growth factor receptor inhibitor, erlotinib, was delivered specifically to myofibroblasts by a versatile nanoparticle-based system, targeting platelet-derived growth factor receptor-beta uniquely expressed on their surface in the liver. With systemic administration of erlotinib, tumor burden was reduced to 31%, which was further improved to 21% by myofibroblast-targeted delivery even with reduced erlotinib dose (7.3-fold reduction with equivalent erlotinib dose) and less hepatocyte damage. These findings demonstrate a strategy, cell type-specific kinase inhibition, for more effective and safer precision cancer chemoprevention.

MPI depletion enhances O-GlcNAcylation of p53 and suppresses the Warburg effect.

Rapid cellular proliferation in early development and cancer depends on glucose metabolism to fuel macromolecule biosynthesis. Metabolic enzymes are presumed regulators of this glycolysis-driven metabolic program, known as the Warburg effect; however, few have been identified. We uncover a previously unappreciated role for Mannose phosphate isomerase (MPI) as a metabolic enzyme required to maintain Warburg metabolism in zebrafish embryos and in both primary and malignant mammalian cells. The functional consequences of MPI loss are striking: glycolysis is blocked and cells die. These phenotypes are caused by induction of p53 and accumulation of the glycolytic intermediate fructose 6-phosphate, leading to engagement of the hexosamine biosynthetic pathway (HBP), increased O-GlcNAcylation, and p53 stabilization. Inhibiting the HBP through genetic and chemical methods reverses p53 stabilization and rescues the Mpi-deficient phenotype. This work provides mechanistic evidence by which MPI loss induces p53, and identifies MPI as a novel regulator of p53 and Warburg metabolism.

BET N-terminal bromodomain inhibition selectively blocks Th17 cell differentiation and ameliorates colitis in mice.

T-helper 17 (Th17) cells have important functions in adaptor immunity and have also been implicated in inflammatory disorders. The bromodomain and extraterminal domain (BET) family proteins regulate gene transcription during lineage-specific differentiation of naïve CD4+ T cells to produce mature T-helper cells. Inhibition of acetyl-lysine binding of the BET proteins by pan-BET bromodomain (BrD) inhibitors, such as JQ1, broadly affects differentiation of Th17, Th1, and Th2 cells that have distinct immune functions, thus limiting their therapeutic potential. Whether these BET proteins represent viable new epigenetic drug targets for inflammatory disorders has remained an unanswered question. In this study, we report that selective inhibition of the first bromodomain of BET proteins with our newly designed small molecule MS402 inhibits primarily Th17 cell differentiation with a little or almost no effect on Th1 or Th2 and Treg cells. MS402 preferentially renders Brd4 binding to Th17 signature gene loci over those of housekeeping genes and reduces Brd4 recruitment of p-TEFb to phosphorylate and activate RNA polymerase II for transcription elongation. We further show that MS402 prevents and ameliorates T-cell transfer-induced colitis in mice by blocking Th17 cell overdevelopment. Thus, selective pharmacological modulation of individual bromodomains likely represents a strategy for treatment of inflammatory bowel diseases.

Structure-Guided Discovery of Selective Antagonists for the Chromodomain of Polycomb Repressive Protein CBX7.

The chromobox 7 (CBX7) protein of the polycomb repressive complex 1 (PRC1) functions to repress transcription of tumor suppressor p16 (INK4a) through long noncoding RNA, ANRIL (antisense noncoding RNA in the INK4 locus) directed chromodomain (ChD) binding to trimethylated lysine 27 of histone H3 (H3K27me3), resulting in chromatin compaction at the INK4a/ARF locus. In this study, we report structure-guided discovery of two distinct classes of small-molecule antagonists for the CBX7ChD. Our Class A compounds, a series including analogues of the previously reported MS452, inhibit CBX7ChD/methyl-lysine binding by occupying the H3K27me3 peptide binding site, whereas our Class B compound, the newly discovered MS351, appears to inhibit H3K27me3 binding when CBX7ChD is bound to RNA. Our crystal structure of the CBX7ChD/MS351 complex reveals the molecular details of ligand recognition by the aromatic cage residues that typically engage in methyl-lysine binding. We further demonstrate that MS351 effectively induces transcriptional derepression of CBX7 target genes, including p16 (INK4a) in mouse embryonic stem cells and human prostate cancer PC3 cells. Thus, MS351 represents a new class of ChD antagonists that selectively targets the biologically active form of CBX7 of the PRC1 in long noncoding RNA- and H3K27me3-directed gene transcriptional repression.

Novel 4-substituted-N,N-dimethyltetrahydronaphthalen-2-amines: synthesis, affinity, and in silico docking studies at serotonin 5-HT2-type and histamine H1 G protein-coupled receptors.

Syntheses were undertaken of derivatives of (2S,4R)-(-)-trans-4-phenyl-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (4-phenyl-2-dimethylaminotetralin, PAT), a stereospecific agonist at the serotonin 5-HT2C G protein-coupled receptor (GPCR), with inverse agonist activity at 5-HT2A and 5-HT2B GPCRs. Molecular changes were made at the PAT C(4)-position, while preserving N,N-dimethyl substitution at the 2-position as well as trans-stereochemistry, structural features previously shown to be optimal for 5-HT2 binding. Affinities of analogs were determined at recombinant human 5-HT2 GPCRs in comparison to the phylogenetically closely-related histamine H1 GPCR, and in silico ligand docking studies were conducted at receptor molecular models to help interpret pharmacological results and guide future ligand design. In most cases, C(4)-substituted PAT analogs exhibited the same stereoselectivity ([-]-trans>[+]-trans) as the parent PAT across 5-HT2 and H1 GPCRs, albeit, with variable receptor selectivity. 4-(4'-substituted)-PAT analogs, however, demonstrated reversed stereoselectivity ([2S,4R]-[+]-trans>[2S,4R]-[-]-trans), with absolute configuration confirmed by single X-ray crystallographic data for the 4-(4'-Cl)-PAT analog. Pharmacological affinity results and computational results herein support further PAT drug development studies and provide a basis for predicting and interpreting translational results, including, for (+)-trans-4-(4'-Cl)-PAT and (-)-trans-4-(3'-Br)-PAT that were previously shown to be more potent and efficacious than their corresponding enantiomers in rodent models of psychoses, psychostimulant-induced behaviors, and compulsive feeding ('binge-eating').

Selective chemical modulation of gene transcription favors oligodendrocyte lineage progression.

Lysine acetylation regulates gene expression through modulating protein-protein interactions in chromatin. Chemical inhibition of acetyl-lysine binding bromodomains of the major chromatin regulators BET (bromodomain and extraterminal domain) proteins has been shown to effectively block cell proliferation in cancer and inflammation. However, whether selective inhibition of individual BET bromodomains has distinctive functional consequences remains only partially understood. In this study, we show that selective chemical inhibition of the first bromodomain of BET proteins using our small-molecule inhibitor, Olinone, accelerated the progression of mouse primary oligodendrocyte progenitors toward differentiation, whereas inhibition of both bromodomains of BET proteins hindered differentiation. This effect was target specific, as it was not detected in cells treated with inactive analogs and independent of any effect on proliferation. Therefore, selective chemical modulation of individual bromodomains, rather than use of broad-based inhibitors, may enhance regenerative strategies in disorders characterized by myelin loss such as aging and neurodegeneration.

Ionic liquids based on azolate anions.

Compartmentalized molecular level design of new energetic materials based on energetic azolate anions allows for the examination of the effects of both cation and anion on the physiochemical properties of ionic liquids. Thirty one novel salts were synthesized by pairing diverse cations (tetraphenylphosphonium, ethyltriphenylphosphonium, N-phenyl pyridinium, 1-butyl-3-methylimidazolium, tetramethyl-, tetraethyl-, and tetrabutylammonium) with azolate anions (5-nitrobenzimidazolate, 5-nitrobenzotriazolate, 3,5-dinitro-1,2,4-triazolate, 2,4-dinitroimidazolate, 4-nitro-1,2,3-triazolate, 4,5-dinitroimidazolate, 4,5-dicyanoimidazolate, 4-nitroimidazolate, and tetrazolate). These salts have been characterized by DSC, TGA, and single crystal X-ray crystallography. The azolates in general are surprisingly stable in the systems explored. Ionic liquids were obtained with all combinations of the 1-butyl-3-methylimidazolium cation and the heterocyclic azolate anions studied, and with several combinations of tetraethyl- or tetrabutylammonium cations and the azolate anions. Favorable structure-property relationships were most often achieved when changing from 4- and 4,5-disubstituted anions to 3,5- and 2,4-disubstituted anions. The most promising anion for use in energetic ionic liquids of those studied here, was 3,5-dinitro-1,2,4-triazolate, based on its contributions to the entire set of target properties.

New Approach to 4-Phenyl-ß-aminotetralin from 4-(3-Halophenyl)tetralen-2-ol Phenylacetate.

Mixed trifluoroacetyl phenylacetyl anhydride and 3-halostyrenes (fluoro, chloro, and bromo) or vinylcycloalkanes (cyclohexyl, cyclooctyl), undergo cascade Friedel-Crafts cycli-acylalkylation, enolization, and O-acylation to give 4-substituted tetralen-2-ol phenylacetates, without additional solvent in good yields. Base alcoholysis of 4-phenyltetralen-2-ol phenylacetate reveals the tetral-2-one for asymmetric transfer hydrogenation. Bromophenyltetralen-2-ol phenylacetate undergoes Suzuki coupling, and provides a short route to trans-4-phenyl-ß-aminotetralin.

Inhibitors of Trypanosoma cruzi trypanothione reductase revealed by virtual screening and parallel synthesis.

In an approach to discover new inhibitors of trypanothione reductase from Trypanosoma cruzi, the causative agent of Chagas' disease, a virtual high-throughput screening was performed. Two structurally new types of inhibitors emerged, the antimicrobial chlorhexidine {1,1'-hexamethylenebis[5-(4-chlorophenyl)biguanide]}, a linear competitive inhibitor (K(i) = 2 +/- 1 microM), and a piperidine derivative acting as mixed inhibitor (K(i) = 6.2 +/- 2 microM and K(i)' = 8.5 +/- 2 microM). Neither compound interferes with human glutathione reductase. Based on chlorhexidine, different series of compounds were synthesized and studied as inhibitors of T. cruzi trypanothione reductase. Most efficient derivatives were three bis(amidines) showing mixed type inhibition with K(i,slope) and K(i,int) values of 2-5 microM and 16-47 microM, respectively. Although these compounds did not exert an improved inhibitory potency compared to chlorhexidine, the change from competitive to mixed-type inhibition is advantageous, since substrate accumulation does not overcome inhibition. Remarkably, all three derivatives carried two copies of an identical 2-methoxy-4-methyl-1-(phenylmethoxy)benzene substituent.