Design, synthesis and cellular characterization of a new class of IPMK kinase inhibitors.

Many genetic studies have established the kinase activity of inositol phosphate multikinase (IPMK) is required for the synthesis of higher-order inositol phosphate signaling molecules, the regulation of gene expression and control of the cell cycle. These genetic studies await orthogonal validation by specific IPMK inhibitors, but no such inhibitors have been synthesized. Here, we report complete chemical synthesis, cellular characterization, structure-activity relationships and rodent pharmacokinetics of a novel series of highly potent IPMK inhibitors.

Dysregulating PHO Signaling via the CDK Machinery Differentially Impacts Energy Metabolism, Calcineurin Signaling, and Virulence in Cryptococcus neoformans.

Fungal pathogens uniquely regulate phosphate homeostasis via the cyclin-dependent kinase (CDK) signaling machinery of the phosphate acquisition (PHO) pathway (Pho85 kinase-Pho80 cyclin-CDK inhibitor Pho81), providing drug-targeting opportunities. Here, we investigate the impact of a PHO pathway activation-defective Cryptococcus neoformans mutant (Δ) and a constitutively activated PHO pathway mutant (Δ) on fungal virulence. Irrespective of phosphate availability, the PHO pathway was derepressed in Δ with all phosphate acquisition pathways upregulated and much of the excess phosphate stored as polyphosphate (polyP).

TNP Analogues Inhibit the Virulence Promoting IP Kinase Arg1 in the Fungal Pathogen .

New antifungals with unique modes of action are urgently needed to treat the increasing global burden of invasive fungal infections. The fungal inositol polyphosphate kinase (IPK) pathway, comprised of IPKs that convert IP to IP, provides a promising new target due to its impact on multiple, critical cellular functions and, unlike in mammalian cells, its lack of redundancy. Nearly all IPKs in the fungal pathway are essential for virulence, with IP kinase (IPK) the most critical.

Inositol polyphosphate-protein interactions: Implications for microbial pathogenicity.

Inositol polyphosphates (IPs) and inositol pyrophosphates (PP-IPs) regulate diverse cellular processes in eukaryotic cells. IPs and PP-IPs are highly negatively charged and exert their biological effects by interacting with specific protein targets. Studies performed predominantly in mammalian cells and model yeasts have shown that IPs and PP-IPs modulate target function through allosteric regulation, by promoting intra- and intermolecular stabilization and, in the case of PP-IPs, by donating a phosphate from their pyrophosphate (PP) group to the target protein.

IP-SPX Domain Interaction Controls Fungal Virulence by Stabilizing Phosphate Signaling Machinery.

In the human-pathogenic fungus , the inositol polyphosphate signaling pathway is critical for virulence. We recently demonstrated the key role of the inositol pyrophosphate IP (isomer 5-PP-IP) in driving fungal virulence; however, the mechanism of action remains elusive. Using genetic and biochemical approaches, and mouse infection models, we show that IP synthesized by Kcs1 regulates fungal virulence by binding to a conserved lysine surface cluster in the SPX domain of Pho81.

Calcium Binding Protein Ncs1 Is Calcineurin Regulated in Cryptococcus neoformans and Essential for Cell Division and Virulence.

Intracellular calcium (Ca) is crucial for signal transduction in , the major cause of fatal fungal meningitis. The calcineurin pathway is the only Ca-requiring signaling cascade implicated in cryptococcal stress adaptation and virulence, with Ca binding mediated by the EF-hand domains of the Ca sensor protein calmodulin. In this study, we identified the cryptococcal ortholog of neuronal calcium sensor 1 (Ncs1) as a member of the EF-hand superfamily.

Monitoring Glycolysis and Respiration Highlights Metabolic Inflexibility of .

is a human fungal pathogen that adapts its metabolism to cope with limited oxygen availability, nutrient deprivation and host phagocytes. To gain insight into cryptococcal metabolism, we optimized a protocol for the Seahorse Analyzer, which measures extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as indications of glycolytic and respiratory activities. In doing so we achieved effective immobilization of encapsulated cryptococci, established Rotenone/Antimycin A and 2-deoxyglucose as effective inhibitors of mitochondrial respiration and glycolysis, respectively, and optimized a microscopy-based method of data normalization.

Fungal Kinases With a Sweet Tooth: Pleiotropic Roles of Their Phosphorylated Inositol Sugar Products in the Pathogenicity of Present Novel Drug Targeting Opportunities.

Invasive fungal pathogens cause more than 300 million serious human infections and 1.6 million deaths per year. A clearer understanding of the mechanisms by which these fungi cause disease is needed to identify novel targets for urgently needed therapies.

The PHO signaling pathway directs lipid remodeling in Cryptococcus neoformans via DGTS synthase to recycle phosphate during phosphate deficiency.

The phosphate sensing and acquisition (PHO) pathway of Cryptococcus neoformans is essential for growth in phosphate-limiting conditions and for dissemination of infection in a mouse model. Its key transcription factor, Pho4, regulates expression of genes controlling the acquisition of phosphate from both external and cellular sources. One such gene, BTA1, is highly up-regulated during phosphate starvation.

The Early Innate Immune Response to, and Phagocyte-Dependent Entry of, Cryptococcus neoformans Map to the Perivascular Space of Cortical Post-Capillary Venules in Neurocryptococcosis.

The innate immune system is the primary defense against cryptococcal infection, but paradoxically it promotes infection of the central nervous system. We performed a detailed longitudinal study of neurocryptococcosis in normal, chimeric, green fluorescent protein phagocyte-positive mice and phagocyte-depleted mice and interrogated the central nervous system innate immune response to Cryptococcus neoformans H99 using confocal microscopy, histology, flow cytometry, and quantification of brain cytokine/chemokines and fungal burdens. C.

IP kinase Arg1 regulates cell wall homeostasis and surface architecture to promote Cryptococcus neoformans infection in a mouse model.

We previously identified a series of inositol polyphosphate kinases (IPKs), Arg1, Ipk1, Kcs1 and Asp1, in the opportunistic fungal pathogen Cryptococcus neoformans. Using gene deletion analysis, we characterized Arg1, Ipk1 and Kcs1 and showed that they act sequentially to convert IP to PP-IP (IP), a key metabolite promoting stress tolerance, metabolic adaptation and fungal dissemination to the brain. We have now directly characterized the enzymatic activity of Arg1, demonstrating that it is a dual specificity (IP/IP) kinase producing IP.

Changes in glucosylceramide structure affect virulence and membrane biophysical properties of Cryptococcus neoformans.

Fungal glucosylceramide (GlcCer) is a plasma membrane sphingolipid in which the sphingosine backbone is unsaturated in carbon position 8 (C8) and methylated in carbon position 9 (C9). Studies in the fungal pathogen, Cryptococcus neoformans, have shown that loss of GlcCer synthase activity results in complete loss of virulence in the mouse model. However, whether the loss of virulence is due to the lack of the enzyme or to the loss of the sphingolipid is not known.

Pho4 Is Essential for Dissemination of to the Host Brain by Promoting Phosphate Uptake and Growth at Alkaline pH.

Phosphate acquisition by fungi is regulated by the phosphate-sensing and acquisition (PHO) signaling pathway. disseminates from the lung to the brain and is the commonest cause of fungal meningitis worldwide. To investigate the contribution of PHO signaling to cryptococcal dissemination, we characterized a transcription factor knockout strain () defective in phosphate acquisition.

Inositol Polyphosphate Kinases, Fungal Virulence and Drug Discovery.

Opportunistic fungi are a major cause of morbidity and mortality world-wide, particularly in immunocompromised individuals. Developing new treatments to combat invasive fungal disease is challenging given that fungal and mammalian host cells are eukaryotic, with similar organization and physiology. Even therapies targeting unique fungal cell features have limitations and drug resistance is emerging.

Identification of a major IP5 kinase in Cryptococcus neoformans confirms that PP-IP5/IP7, not IP6, is essential for virulence.

Fungal inositol polyphosphate (IP) kinases catalyse phosphorylation of IP3 to inositol pyrophosphate, PP-IP5/IP7, which is essential for virulence of Cryptococcus neoformans. Cryptococcal Kcs1 converts IP6 to PP-IP5/IP7, but the kinase converting IP5 to IP6 is unknown. Deletion of a putative IP5 kinase-encoding gene (IPK1) alone (ipk1Δ), and in combination with KCS1 (ipk1Δkcs1Δ), profoundly reduced virulence in mice.

Fungal Inositol Pyrophosphate IP7 Is Crucial for Metabolic Adaptation to the Host Environment and Pathogenicity.

Unlabelled: Inositol pyrophosphates (PP-IPs) comprising inositol, phosphate, and pyrophosphate (PP) are essential for multiple functions in eukaryotes. Their role in fungal pathogens has never been addressed. Cryptococcus neoformans is a model pathogenic fungus causing life-threatening meningoencephalitis.

Rapid microscopy and use of vital dyes: potential to determine viability of Cryptococcus neoformans in the clinical laboratory.

Background: Cryptococcus neoformans is the commonest cause of fungal meningitis, with a substantial mortality despite appropriate therapy. Quantitative culture of cryptococci in cerebrospinal fluid (CSF) during antifungal therapy is of prognostic value and has therapeutic implications, but is slow and not practicable in many resource-poor countries.

Methods: We piloted two rapid techniques for quantifying viable cryptococci using mixtures of live and heat-killed cryptococci cultured in vitro: (i) quantitative microscopy with exclusion staining using trypan blue dye, and (ii) flow cytometry, using the fluorescent dye 2'-7'-Bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM).

Identification of Aph1, a phosphate-regulated, secreted, and vacuolar acid phosphatase in Cryptococcus neoformans.

Unlabelled: Cryptococcus neoformans strains isolated from patients with AIDS secrete acid phosphatase, but the identity and role of the enzyme(s) responsible have not been elucidated. By combining a one-dimensional electrophoresis step with mass spectrometry, a canonically secreted acid phosphatase, CNAG_02944 (Aph1), was identified in the secretome of the highly virulent serotype A strain H99. We created an APH1 deletion mutant (Δaph1) and showed that Δaph1-infected Galleria mellonella and mice survived longer than those infected with the wild type (WT), demonstrating that Aph1 contributes to cryptococcal virulence.

Phospholipase C of Cryptococcus neoformans regulates homeostasis and virulence by providing inositol trisphosphate as a substrate for Arg1 kinase.

Phospholipase C (PLC) of Cryptococcus neoformans (CnPlc1) is crucial for virulence of this fungal pathogen. To investigate the mechanism of CnPlc1-mediated signaling, we established that phosphatidylinositol 4,5-bisphosphate (PIP(2)) is a major CnPlc1 substrate, which is hydrolyzed to produce inositol trisphosphate (IP(3)). In Saccharomyces cerevisiae, Plc1-derived IP(3) is a substrate for the inositol polyphosphate kinase Arg82, which converts IP(3) to more complex inositol polyphosphates.

The Crz1/Sp1 transcription factor of Cryptococcus neoformans is activated by calcineurin and regulates cell wall integrity.

Cryptococcus neoformans survives host temperature and regulates cell wall integrity via a calcium-dependent phosphatase, calcineurin. However, downstream effectors of C. neoformans calcineurin are largely unknown.

Miltefosine induces apoptosis-like cell death in yeast via Cox9p in cytochrome c oxidase.

Miltefosine has antifungal properties and potential for development as a therapeutic for invasive fungal infections. However, its mode of action in fungi is poorly understood. We demonstrate that miltefosine is rapidly incorporated into yeast, where it penetrates the mitochondrial inner membrane, disrupting mitochondrial membrane potential and leading to an apoptosis-like cell death.