Antifungal Activity of Gepinacin Scaffold Glycosylphosphatidylinositol Anchor Biosynthesis Inhibitors with Improved Metabolic Stability.

The glycosylphosphatidylinositol anchor biosynthesis inhibitor gepinacin demonstrates broad-spectrum antifungal activity and negligible mammalian toxicity in culture but is metabolically labile. The stability and bioactivity of 39 analogs were tested in vitro to identify LCUT-8, a stabilized lead with increased potency and promising single-dose pharmacokinetics. Unfortunately, no antifungal activity was seen at the maximum dosing achievable in a neutropenic rabbit model. Nevertheless, structure-activity relationships identified here suggest strategies to further improve compound potency, solubility, and stability.

Structural basis for species-selective targeting of Hsp90 in a pathogenic fungus.

New strategies are needed to counter the escalating threat posed by drug-resistant fungi. The molecular chaperone Hsp90 affords a promising target because it supports survival, virulence and drug-resistance across diverse pathogens. Inhibitors of human Hsp90 under development as anticancer therapeutics, however, exert host toxicities that preclude their use as antifungals. Seeking a route to species-selectivity, we investigate the nucleotide-binding domain (NBD) of Hsp90 from the most common human fungal pathogen, Candida albicans. Here we report structures for this NBD alone, in complex with ADP or in complex with known Hsp90 inhibitors. Encouraged by the conformational flexibility revealed by these structures, we synthesize an inhibitor with >25-fold binding-selectivity for fungal Hsp90 NBD. Comparing co-crystals occupied by this probe vs. anticancer Hsp90 inhibitors revealed major, previously unreported conformational rearrangements. These insights and our probe's species-selectivity in culture support the feasibility of targeting Hsp90 as a promising antifungal strategy.

Handheld versus conventional vascular ultrasound for assessing carotid artery plaque.

BACKGROUND: Coronary Artery Disease (CAD) is the leading cause of death worldwide. Ultrasound-detected carotid plaque has been previously shown to predict significant CAD. Despite this evidence, carotid plaque assessment has not been implemented in cardiac risk screening, likely due to the cost associated with a formal carotid ultrasound examination. This study sought to determine whether a handheld vascular ultrasound device could be used as an accurate point-of-care imaging tool for the assessment of carotid artery plaque. METHODS: We performed a focused vascular ultrasound of the carotid arteries of 200 patients referred for coronary angiography using a handheld ultrasound device as well as a full-size conventional ultrasound system. For each participant, the maximum plaque height (MPH) and total plaque area (TPA) of the carotid artery bulbs were measured. RESULTS: Carotid plaque assessment using the handheld device was comparable to that of the conventional ultrasound system. We found a good correlation and no relevant bias between handheld and conventional ultrasound systems in measuring MPH (r = 0.84, p < 0.0001) and TPA (r = 0.94, p < 0.0001). Furthermore, there was good inter-rater reliability for online and offline measurements of MPH and offline measurements of TPA by handheld ultrasound (0.79, 0.76 and 0.85, respectively). CONCLUSIONS: This study demonstrates that a focused ultrasound of the carotid artery using a handheld device can be used to accurately measure MPH and TPA. This protocol has the potential to provide an expedited point-of-care assessment of carotid plaque.

Inhibiting mitochondrial phosphate transport as an unexploited antifungal strategy.

The development of effective antifungal therapeutics remains a formidable challenge because of the close evolutionary relationship between humans and fungi. Mitochondrial function may present an exploitable vulnerability because of its differential utilization in fungi and its pivotal roles in fungal morphogenesis, virulence, and drug resistance already demonstrated by others. We now report mechanistic characterization of ML316, a thiohydantoin that kills drug-resistant Candida species at nanomolar concentrations through fungal-selective inhibition of the mitochondrial phosphate carrier Mir1. Using genetic, biochemical, and metabolomic approaches, we established ML316 as the first Mir1 inhibitor. Inhibition of Mir1 by ML316 in respiring yeast diminished mitochondrial oxygen consumption, resulting in an unusual metabolic catastrophe marked by citrate accumulation and death. In a mouse model of azole-resistant oropharyngeal candidiasis, ML316 reduced fungal burden and enhanced azole activity. Targeting Mir1 could provide a new, much-needed therapeutic strategy to address the rapidly rising burden of drug-resistant fungal infection.

Sex Differences of the Natriuretic Peptide Polymorphism Associated With Angiographic Coronary Atherosclerosis.

BACKGROUND: Polymorphisms within natriuretic peptide (NP) genes have been associated with clinical outcomes for cardiovascular disease (CVD), but no previous study has compared the effect of these polymorphisms between men and women. This study aimed to investigate the association between single nucleotide polymorphisms (SNPs) in key genes of the NP system and coronary angiographic outcomes, with the focus on the sexual dimorphism in the effects of these SNPs. METHODS: Patients undergoing clinically indicated coronary angiography (n = 513, 328 men and 185 women) were consented and genotyped for NPPA rs5065, NPPB rs198389 and NPR2 rs10758325. Patients were stratified into having normal coronaries, non-obstructive coronary artery disease (CAD) or obstructive CAD, based on the highest stenosis in any epicardial artery. Average luminal narrowing across all four arteries was derived to represent the overall atherosclerotic burden. RESULTS: The frequency of NPPB rs198389 AA genotype was significantly higher in women with obstructive CAD (P = 0.014). The same association was not observed in males. With respect to atherosclerotic burden, an association was found between the AA genotype and average luminal narrowing in women (P = 0.005), but not in men. CONCLUSIONS: The current study identified an association between an SNP of the NPPB gene and coronary atherosclerotic burden through angiographic evidence in women but not in men. These results suggest that B-type natriuretic peptide (BNP) may have more important involvement in the development of CAD in women compared to men, and as such, genotyping of the NPPB gene may serve as a potential biomarker to identify women with high risk for CAD.

Chemical Genomics-Based Antifungal Drug Discovery: Targeting Glycosylphosphatidylinositol (GPI) Precursor Biosynthesis.

Steadily increasing antifungal drug resistance and persistent high rates of fungal-associated mortality highlight the dire need for the development of novel antifungals. Characterization of inhibitors of one enzyme in the GPI anchor pathway, Gwt1, has generated interest in the exploration of targets in this pathway for further study. Utilizing a chemical genomics-based screening platform referred to as the Candida albicans fitness test (CaFT), we have identified novel inhibitors of Gwt1 and a second enzyme in the glycosylphosphatidylinositol (GPI) cell wall anchor pathway, Mcd4. We further validate these targets using the model fungal organism Saccharomyces cerevisiae and demonstrate the utility of using the facile toolbox that has been compiled in this species to further explore target specific biology. Using these compounds as probes, we demonstrate that inhibition of Mcd4 as well as Gwt1 blocks the growth of a broad spectrum of fungal pathogens and exposes key elicitors of pathogen recognition. Interestingly, a strong chemical synergy is also observed by combining Gwt1 and Mcd4 inhibitors, mirroring the demonstrated synthetic lethality of combining conditional mutants of GWT1 and MCD4. We further demonstrate that the Mcd4 inhibitor M720 is efficacious in a murine infection model of systemic candidiasis. Our results establish Mcd4 as a promising antifungal target and confirm the GPI cell wall anchor synthesis pathway as a promising antifungal target area by demonstrating that effects of inhibiting it are more general than previously recognized.

Inhibiting GPI anchor biosynthesis in fungi stresses the endoplasmic reticulum and enhances immunogenicity.

In fungi, the anchoring of proteins to the plasma membrane via their covalent attachment to glycosylphosphatidylinositol (GPI) is essential and thus provides a valuable point of attack for the development of antifungal therapeutics. Unfortunately, studying the underlying biology of GPI-anchor synthesis is difficult, especially in medically relevant fungal pathogens because they are not genetically tractable. Compounding difficulties, many of the genes in this pathway are essential in Saccharomyces cerevisiae. Here, we report the discovery of a new small molecule christened gepinacin (for GPI acylation inhibitor) which selectively inhibits Gwt1, a critical acyltransferase required for the biosynthesis of fungal GPI anchors. After delineating the target specificity of gepinacin using genetic and biochemical techniques, we used it to probe key, therapeutically relevant consequences of disrupting GPI anchor metabolism in fungi. We found that, unlike all three major classes of antifungals in current use, the direct antimicrobial activity of this compound results predominantly from its ability to induce overwhelming stress to the endoplasmic reticulum. Gepinacin did not affect the viability of mammalian cells nor did it inhibit their orthologous acyltransferase. This enabled its use in co-culture experiments to examine Gwt1's effects on host-pathogen interactions. In isolates of Candida albicans, the most common fungal pathogen in humans, exposure to gepinacin at sublethal concentrations impaired filamentation and unmasked cell wall ß-glucan to stimulate a pro-inflammatory cytokine response in macrophages. Gwt1 is a promising antifungal drug target, and gepanacin is a useful probe for studying how disrupting GPI-anchor synthesis impairs viability and alters host-pathogen interactions in genetically intractable fungi.

A rhizosphere fungus enhances Arabidopsis thermotolerance through production of an HSP90 inhibitor.

The molecular chaperone HEAT SHOCK PROTEIN90 (HSP90) is essential for the maturation of key regulatory proteins in eukaryotes and for the response to temperature stress. Earlier, we have reported that fungi living in association with plants of the Sonoran desert produce small molecule inhibitors of mammalian HSP90. Here, we address whether elaboration of the HSP90 inhibitor monocillin I (MON) by the rhizosphere fungus Paraphaeosphaeria quadriseptata affects plant HSP90 and plant environmental responsiveness. We demonstrate that MON binds Arabidopsis (Arabidopsis thaliana) HSP90 and can inhibit the function of HSP90 in lysates of wheat (Triticum aestivum) germ. MON treatment of Arabidopsis seedlings induced HSP101 and HSP70, conserved components of the stress response. Application of MON, or growth in the presence of MON, allowed Arabidopsis wild type but not AtHSP101 knockout mutant seedlings to survive otherwise lethal temperature stress. Finally, cocultivation of P. quadriseptata with Arabidopsis enhanced plant heat stress tolerance. These data demonstrate that HSP90-inhibitory compounds produced by fungi can influence plant growth and responses to the environment.

Phenotypic diversity and altered environmental plasticity in Arabidopsis thaliana with reduced Hsp90 levels.

The molecular chaperone HSP90 aids the maturation of a diverse but select set of metastable protein clients, many of which are key to a variety of signal transduction pathways. HSP90 function has been best investigated in animal and fungal systems, where inhibition of the chaperone has exceptionally diverse effects, ranging from reversing oncogenic transformation to preventing the acquisition of drug resistance. Inhibition of HSP90 in the model plant Arabidopsis thaliana uncovers novel morphologies dependent on normally cryptic genetic variation and increases stochastic variation inherent to developmental processes. The biochemical activity of HSP90 is strictly conserved between animals and plants. However, the substrates and pathways dependent on HSP90 in plants are poorly understood. Progress has been impeded by the necessity of reliance on light-sensitive HSP90 inhibitors due to redundancy in the A. thaliana HSP90 gene family. Here we present phenotypic and genome-wide expression analyses of A. thaliana with constitutively reduced HSP90 levels achieved by RNAi targeting. HSP90 reduction affects a variety of quantitative life-history traits, including flowering time and total seed set, increases morphological diversity, and decreases the developmental stability of repeated characters. Several morphologies are synergistically affected by HSP90 and growth temperature. Genome-wide expression analyses also suggest a central role for HSP90 in the genesis and maintenance of plastic responses. The expression results are substantiated by examination of the response of HSP90-reduced plants to attack by caterpillars of the generalist herbivore Trichoplusia ni. HSP90 reduction potentiates a more robust herbivore defense response. In sum, we propose that HSP90 exerts global effects on the environmental responsiveness of plants to many different stimuli. The comprehensive set of HSP90-reduced lines described here is a vital instrument to further examine the role of HSP90 as a central interface between organism, development, and environment.

Association between completeness of percutaneous coronary revascularization and postprocedure outcomes.

BACKGROUND: Multivessel coronary artery revascularization may be accomplished by percutaneous coronary intervention (PCI) or coronary artery bypass graft surgery (CABG). The importance of complete revascularization is emphasized in the surgical literature, but little is known about its impact on PCI outcomes. This study evaluated multivessel PCI patients to determine the predictors of complete revascularization and the association of complete revascularization with survival, subsequent CABG, and repeat PCI. METHODS: The Alberta Provincial Project for Outcomes Assessment in Coronary Heart Disease (APPROACH) is a clinical data collection and outcome-monitoring initiative capturing all patients undergoing cardiac catheterization and revascularization in the province of Alberta, Canada. Characteristics and long-term outcomes of 1308 patients undergoing multivessel PCI with complete revascularization were compared with those of 648 patients with incomplete revascularization. RESULTS: The significant independent predictors of complete revascularization were pre-PCI Duke jeopardy score, the presence of a total occlusion, year of PCI, age > 65 years, renal failure, and left ventricular function. With a median follow-up time of 3.0 +/- 1.8 years, the adjusted hazard ratio (HR) (95% CI) for the association between complete revascularization and outcome was 0.75 (0.54-1.04) for death, 0.55 (0.37-0.84) for subsequent CABG, and 0.93 (0.65-1.34) for repeat PCI. CONCLUSIONS: Baseline angiographic characteristics and other clinical factors can predict complete revascularization in patients undergoing multivessel PCI. Complete multivessel PCI is associated with reduced need for future CABG, a trend toward better survival, and no difference in repeat PCI.

Current reperfusion strategies for ST elevation myocardial infarction: a Canadian perspective.

The goal of reperfusion therapy for ST elevation myocardial infarction is to achieve prompt and sustained patency of the infarct-related artery. Fibrinolytic therapy is quickly available but it successfully restores flow only in approximately 60% of cases. Percutaneous coronary intervention (PCI) is successful in over 90% of cases but requires timely availability of an experienced interventional team. Although individual trials do not show a clear mortality benefit with PCI, a recent analysis of 23 trials comparing PCI with fibrinolytic therapy showed PCI was associated with improved survival. PCI is also associated with less recurrent ischemia, reinfarction, stroke, target vessel revascularization and a shorter hospital stay. Facilitated PCI attempts to optimize the benefits of pharmacological and mechanical reperfusion by giving a pharmacological agent initially, hoping for reperfusion while an emergency PCI is organized. The literature regarding pharmacological and mechanical reperfusion is reviewed to emphasize the important issues while a future strategy of facilitated PCI is contemplated.

Atrial fibrillation in the pacemaker clinic.

BACKGROUND: Electrocardiographic (ECG) recognition of the underlying rhythm in patients with ventricular pacing can be difficult. Atrial fibrillation (AF) in particular may go unreported. OBJECTIVES: To compare the underlying atrial rhythm determined in the pacemaker clinic with the 12-lead ECG interpretation of the atrial rhythm in those who were continuously paced in the ventricle. It was intended to determine whether long term anticoagulation therapy was related to whether AF was diagnosed before or after pacemaker implantation. METHODS: Pacemaker clinic patients were enrolled if they had a 100% paced ventricular rhythm. The underlying rhythm was determined using pacemaker programming manoeuvres. A 12-lead ECG was recorded on all patients within 10 min of their pacemaker assessment and interpreted by one of the several geographic full-time cardiologists at the centre. All cardiologists were blinded to the results of pacemaker assessment and to the clinical history. RESULTS: Fifty-six patients were enrolled. At the pacemaker clinic, 37 were determined to be in AF and three were in atrial flutter (AFL). Of these 40 patients with AF/AFL, 28 were correctly identified as such on the 12-lead ECG interpretation. Twelve of the 40 were interpreted only as having an 'electronic ventricular pacemaker' (EVP). Sixteen of the 40 patients (40.0%) with AF or AFL were not taking warfarin. Twenty-two of 25 patients with an AF/AFL diagnosis before pacemaker implantation were taking warfarin, compared with two of 15 patients with AF/AFL diagnosis after pacemaker implantation (P<0.0001). CONCLUSION: These results show that the underlying rhythm in patients with ventricular pacing is frequently unrecognized by routine ECG interpretation. This may be of particular importance in the AF/AFL population as a potential contributor to the underuse of warfarin, especially when AF develops after pacemaker implantation. The pacemaker clinic may be in a position to play an important role in the identification of these patients.

HspBP1, an Hsp70 cochaperone, has two structural domains and is capable of altering the conformation of the Hsp70 ATPase domain.

We present here the first structural information for HspBP1, an Hsp70 cochaperone. Using circular dichroism, HspBP1 was determined to be 35% helical. Although HspBP1 is encoded by seven exons, limited proteolysis shows that it has only two structural domains. Domain I, amino acids 1-83, is largely unstructured. Domain II, amino acids 84-359, is predicted to be 43% helical using circular dichroism. Using limited proteolysis we have also shown that HspBP1 association changes the conformation of the ATPase domain of Hsp70. Only domain II of HspBP1 is required to bring about this conformational change. Truncation mutants of HspBP1 were tested for their ability to inhibit the renaturation of luciferase and bind to Hsp70 in reticulocyte lysate. A carboxyl terminal truncation mutant that was slightly longer than domain I was inactive in these assays, but domain II was sufficient to perform both functions. Domain II was less active than full-length HspBP1 in these assays, and addition of amino acids from domain I improved both functions. These studies show that HspBP1 domain II can bind Hsp70, change the conformation of the ATPase domain, and inhibit Hsp70-associated protein folding.

Increased expression of the Hsp70 cochaperone HspBP1 in tumors.

Hsp70 levels are elevated in a number of different tumors. The Hsp70 cochaperone heat shock protein-binding protein 1 (HspBP1) has been shown to bind to Hsp70, inhibit its activity and promote dissociation of nucleotide from the Hsp70 ATPase domain. The purpose of this study was to determine if the levels of HspBP1 are altered in tumor cells. In this report, we show that HspBP1 levels are elevated in two mouse tumor models, 3LL cells (Lewis Lung carcinoma) and neuroblastoma tumors. The amounts of HspBP1 and Hsp70 in selected tissues, tumors and a rabbit reticulocyte lysate were determined using Western blots. It was found that the molar ratio of these two proteins was within a small range (0.21-0.42) in the normal and tumor tissues examined. This ratio was considerably below the HspBP1 to Hsp70 ratio of 4.0 needed for 50% inhibition of Hsp70-mediated refolding of a partially denatured protein in rabbit reticulocyte lysate. The ratio of HspBP1 to Hsp70 in these tissues is too low to inhibit Hsp70 globally in the cell, but is high enough to provide a pool of HspBP1 that could inhibit Hsp70 in a localized fashion. These studies have shown that HspBP1 is elevated in the tumors examined and therefore could be a new cancer marker.

HspBP1, a homologue of the yeast Fes1 and Sls1 proteins, is an Hsc70 nucleotide exchange factor.

The yeast FES1 and SLS1 genes encode conserved nucleotide exchange factors that act on the cytoplasmic and endoplasmic reticulum luminal Hsp70s, Ssa1p and BiP, respectively. We report here that mammalian HspBP1 is homologous to Fes1p and that HspBP1 promotes nucleotide dissociation from both Ssa1p and mammalian Hsc70. In contrast, Fes1p inefficiently strips nucleotide from mammalian Hsc70, and unlike HspBP1 does not inhibit chaperone-mediated protein refolding in vitro. Together, our data indicate that HspBP1 is a member of this new class of nucleotide exchange factors that exhibit varying degrees of compartment and species specificity.