Nature's Own Pharmacy: Mushroom-Based Chemical Scaffolds and Their Therapeutic Implications.

Mushrooms are new potential sources of valuable medicines, long neglected because of difficulties experienced in their cultivation. There is a large variety of medicinal mushrooms which possess significant therapeutic properties and are used as medications for various diseases because they contain several novel highly bioactive components. Medicinal mushrooms can be identified based on their morphology, size, mass, and the color of the stalk, cap and spore, and attachment to the stalk. Medicinal mushrooms possess a variety of important biological activities and are used as antioxidants, hepatoprotectors, anticancer, antidiabetic, anti-inflammatory, antiaging, antiviral, antiparasitic, and antimicrobial agents, among others. This review provides a basic overview of the chemical scaffolds present in mushrooms and their therapeutic implications in the human body.

Sulfatase 2 Is Associated with Steroid Resistance in Childhood Nephrotic Syndrome.

Glucocorticoid (GC) resistance complicates the treatment of ~10-20% of children with nephrotic syndrome (NS), yet the molecular basis for resistance remains unclear. We used RNAseq analysis and in silico algorithm-based approaches on peripheral blood leukocytes from 12 children both at initial NS presentation and after ~7 weeks of GC therapy to identify a 12-gene panel able to differentiate steroid resistant NS (SRNS) from steroid-sensitive NS (SSNS). Among this panel, subsequent validation and analyses of one biologically relevant candidate, sulfatase 2 (SULF2), in up to a total of 66 children, revealed that both SULF2 leukocyte expression and plasma arylsulfatase activity Post/Pre therapy ratios were greater in SSNS vs. SRNS. However, neither plasma SULF2 endosulfatase activity (measured by VEGF binding activity) nor plasma VEGF levels, distinguished SSNS from SRNS, despite VEGF's reported role as a downstream mediator of SULF2's effects in glomeruli. Experimental studies of NS-related injury in both rat glomeruli and cultured podocytes also revealed decreased SULF2 expression, which were partially reversible by GC treatment of podocytes. These findings together suggest that SULF2 levels and activity are associated with GC resistance in NS, and that SULF2 may play a protective role in NS via the modulation of downstream mediators distinct from VEGF.

Phosphatidylinositol 4-phosphate is a major source of GPCR-stimulated phosphoinositide production.

Phospholipase C (PLC) enzymes hydrolyze the plasma membrane (PM) lipid phosphatidylinositol 4,5-bisphosphate (PI4,5P2) to generate the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG) in response to receptor activation in almost all mammalian cells. We previously found that stimulation of G protein-coupled receptors (GPCRs) in cardiac cells leads to the PLC-dependent hydrolysis of phosphatidylinositol 4-phosphate (PI4P) at the Golgi, a process required for the activation of nuclear protein kinase D (PKD) during cardiac hypertrophy. We hypothesized that GPCR-stimulated PLC activation leading to direct PI4P hydrolysis may be a general mechanism for DAG production. We measured GPCR activation-dependent changes in PM and Golgi PI4P pools in various cells using GFP-based detection of PI4P. Stimulation with various agonists caused a time-dependent reduction in PI4P-associated, but not PI4,5P2-associated, fluorescence at the Golgi and PM. Targeted depletion of PI4,5P2 from the PM before GPCR stimulation had no effect on the depletion of PM or Golgi PI4P, total inositol phosphate (IP) production, or PKD activation. In contrast, acute depletion of PI4P specifically at the PM completely blocked the GPCR-dependent production of IPs and activation of PKD but did not change the abundance of PI4,5P2 Acute depletion of Golgi PI4P had no effect on these processes. These data suggest that most of the PM PI4,5P2 pool is not involved in GPCR-stimulated phosphoinositide hydrolysis and that PI4P at the PM is responsible for the bulk of receptor-stimulated phosphoinositide hydrolysis and DAG production.

Small heat shock protein speciation: novel non-canonical 44 kDa HspB5-related protein species in rat and human tissues.

When analyzing small stress proteins of rat and human tissues by electrophoretic methods followed by western blotting, and using the anti-HspB1/anti-HspB5 antibody clone 8A7, we unexpectedly found a protein with a molecular mass of ~44 kDa. On two-dimensional gels, this protein resolved into four distinct species. Electrophoretic and immunological evidence suggests that this 44 kDa protein is a derivative of HspB5, most likely a covalently linked HspB5 dimer. This HspB5-like 44 kDa protein (HspB5L-P44) is particularly abundant in rat heart, brain, and renal cortex and glomeruli. HspB5L-P44 was also found in human brains, including those from patients with Alexander disease, a condition distinguished by cerebral accumulation of HspB5. Gray matter of such a patient contained an elevated amount of HspB5L-P44. A spatial model of structurally ordered dimeric HspB5 α-crystallin domains reveals the exposed and adjacent position of the two peptide segments homologous to the HspB1-derived 8A7 antigen determinant peptide (epitope). This explains the observed extraordinary high avidity of the 8A7 antibody towards HspB5L-P44, as opposed to commonly used HspB5-specific antibodies which recognize other epitopes. This scenario also explains the remarkable fact that no previous study reported the existence of HspB5L-P44 species. Exposure of rat endothelial cells to UV light, an oxidative stress condition, temporarily increased HspB5L-P44, suggesting physiological regulation of the dimerization. The existence of HspB5L-P44 supports the protein speciation discourse and fits to the concept of the protein code, according to which the expression of a given gene is reflected only by the complete set of the derived protein species.

DGKE variants cause a glomerular microangiopathy that mimics membranoproliferative GN.

Renal microangiopathies and membranoproliferative GN (MPGN) can manifest similar clinical presentations and histology, suggesting the possibility of a common underlying mechanism in some cases. Here, we performed homozygosity mapping and whole exome sequencing in a Turkish consanguineous family and identified DGKE gene variants as the cause of a membranoproliferative-like glomerular microangiopathy. Furthermore, we identified two additional DGKE variants in a cohort of 142 unrelated patients diagnosed with membranoproliferative GN. This gene encodes the diacylglycerol kinase DGKε, which is an intracellular lipid kinase that phosphorylates diacylglycerol to phosphatidic acid. Immunofluorescence confocal microscopy demonstrated that mouse and rat Dgkε colocalizes with the podocyte marker WT1 but not with the endothelial marker CD31. Patch-clamp experiments in human embryonic kidney (HEK293) cells showed that DGKε variants affect the intracellular concentration of diacylglycerol. Taken together, these results not only identify a genetic cause of a glomerular microangiopathy but also suggest that the phosphatidylinositol cycle, which requires DGKE, is critical to the normal function of podocytes.

Marker expression, behaviors, and responses vary in different lines of conditionally immortalized cultured podocytes.

The state-of-the-art cultured podocyte is conditionally immortalized by expression of a temperature-sensitive mutant of the SV40 large-T antigen. These cultures proliferate at 33°C and differentiate at 37°C into arborized cells that more closely resemble in vivo podocytes. However, the degree of resemblance remains controversial. In this study, several parameters were measured in podocyte cell lines derived from mouse (JR, KE), human (MS), and rat (HK). In all lines, the quantities of NEPH1 and podocin proteins and NEPH1 and SYNPO mRNAs were comparable to glomeruli, while synaptopodin and nephrin proteins and NPHS1 and NPHS2 mRNAs were <5% of glomerular levels. Expression of Wilms' tumor-1 (WT1) mRNA in mouse lines was comparable to glomeruli, but rat and human lines expressed little WT1. Undifferentiated human and mouse lines had similar proliferation rates that decreased after differentiation, while the rate in rat cells remained constant. The motility of different lines varied as measured by both general motility and wound-healing assays. The toxicity of puromycin aminonucleoside was MS ∼ JR >> KE, and of doxorubicin was JR ∼ KE > MS, while HK cells were almost unaffected. Process formation was largely a result of contractile action after formation of lamellipodia. These findings demonstrate dramatic differences in marker expression, response to toxins, and motility between lines of podocytes from different species and even between similarly-derived mouse lines.

Inhibition of the protein kinase MK-2 protects podocytes from nephrotic syndrome-related injury.

While mitogen-activated protein kinase (MAPK) activation has been implicated in the pathogenesis of various glomerular diseases, including nephrotic syndrome (NS), its specific role in podocyte injury is not known. We hypothesized that MK-2, a downstream substrate of p38 MAPK, mediates the adverse effects of this pathway and that inhibition of MK-2 would protect podocytes from NS-related injury. Using cultured podocytes, we analyzed 1) the roles of MK-2 and p38 MAPK in puromycin aminonucleoside (PAN)-induced podocyte injury; 2) the ability of specific MK-2 and p38 MAPK inhibitors to protect podocytes against injury; 3) the role of serum albumin, known to induce podocyte injury, in activating p38 MAPK/MK-2 signaling; and 4) the role of p38 MAPK/MK-2 signaling in the expression of Cox-2, an enzyme associated with podocyte injury. Treatment with protein kinase inhibitors specific for both MK-2 (C23, a pyrrolopyridine-type compound) or p38 MAPK (SB203580) reduced PAN-induced podocyte injury and actin cytoskeletal disruption. Both inhibitors reduced baseline podocyte p38 MAPK/MK-2 signaling, as measured by the degree of phosphorylation of HSPB1, a downstream substrate of MK-2, but exhibited disparate effects on upstream signaling. Serum albumin activated p38 MAPK/MK-2 signaling and induced Cox-2 expression, and these responses were blocked by both inhibitors. Given the critical importance of podocyte injury to both NS and other progressive glomerular diseases, these data suggest an important role for p38 MAPK/MK-2 signaling in podocyte injury and identify MK-2 inhibition as a promising potential therapeutic strategy to protect podocytes in various glomerular diseases.

Indazole derivatives as novel bradykinin B1 receptor antagonists.

A new class of indazole-derived bradykinin B(1) antagonists and their structure-activity relationships (SAR) is reported. A number of compounds were found to have low-nanomolar affinity for the human B(1) receptor and possess acceptable P-gp and pharmacokinetics properties.

Dose- and time-dependent glucocorticoid receptor signaling in podocytes.

Glucocorticoids (GC) are the primary therapy for idiopathic nephrotic syndrome (NS). Recent evidence has identified glomerular podocytes as a potential site of GC action in this disease. The objectives of this study were to determine the presence of key components of the glucocorticoid receptor (GR) complex and the functionality of this signaling pathway in podocytes and to explore potential opportunities for manipulation of GC responsiveness. Here, we show that cultured murine podocytes express key components of the GR complex, including the GR, heat shock protein 90, and the immunophilins FKBP51 and FKBP52. The functionality of GR-mediated signaling was verified by measuring several GC (dexamethasone)-induced responses, including 1) increases in mRNA and protein levels of selected GC-regulated genes (FKBP51, phenol sulfotransferase 1, αB-crystallin); 2) downregulation of the GR protein; 3) increased phosphorylation of the GR; and 4) translocation of the GR into the nuclear fraction. Dexamethasone-induced phosphorylation and downregulation of GR protein were also demonstrated in isolated rat glomeruli. Podocyte gene expression in response to dexamethasone was regulated at both the transcriptional and posttranscriptional levels, the latter also including protein degradation. Short-term, high-dose GC treatment resulted in similar changes in gene expression and GR phosphorylation to that of long-term, low-dose GC treatment, thus providing a molecular rationale for the known efficacy of pulse GC therapy in NS. Induction of FKBP51 and downregulation of the GR represent negative feedback mechanisms that can potentially be exploited to improve clinical GC efficacy. Collectively, these findings demonstrate the presence of key molecular components of the GR signaling pathway and its functionality in podocytes and identify novel opportunities for improving clinical GC efficacy in the treatment of NS.

Bradykinin B1 receptor antagonists: an alpha-hydroxy amide with an improved metabolism profile.

A series of carbo- and heterocyclic alpha-hydroxy amide-derived bradykinin B1 antagonists was prepared and evaluated. A 4,4-difluorocyclohexyl alpha-hydroxy amide was incorporated along with a 2-methyl tetrazole in lieu of an oxadiazole to afford a suitable compound with good pharmacokinetic properties, CNS penetration, and clearance by multiple metabolic pathways.

A new class of bradykinin B1 receptor antagonists with high oral bioavailability and minimal PXR activity.

The design and synthesis of a novel class of human bradykinin B1 antagonists featuring difluoroethyl ether and isoxazole carboxamide moieties are disclosed. Compound 7g displayed excellent pharmacokinetic properties, efficient ex vivo receptor occupancy, and low potential for P450 induction via PXR activation.

Proline bis-amides as potent dual orexin receptor antagonists.

A series of OX(2)R/OX(1)R dual orexin antagonists was prepared based on a proline bis-amide identified as a screening lead. Through a combination of classical and library synthesis, potency enhancing replacements for both amide portions were discovered. N-methylation of the benzimidazole moiety within the lead structure significantly reduced P-gp susceptibility while increasing potency, giving rise to good brain penetration. A compound from this series has demonstrated in vivo central activity when dosed peripherally in a pharmacodynamic model of orexin activity.

The podocyte and diabetes mellitus: is the podocyte the key to the origins of diabetic nephropathy?

PURPOSE OF REVIEW: Podocyte injury plays a key role in the development of diabetic nephropathy. This review discusses recent advances in our understanding of mechanisms of podocyte injury in diabetes mellitus and the associated alterations in the function of the glomerular filtration barrier. RECENT FINDINGS: The effects of hyperglycemia on critical podocyte parameters including cell-cell interactions, attachment to the glomerular basement membrane, and podocyte apoptosis have been determined in both cell culture and in-vivo models of diabetes mellitus. The podocyte has also been identified as a target of action for insulin and growth hormone, hormones with significant roles in the altered homeostasis of diabetes mellitus. SUMMARY: Understanding the cellular and molecular basis for changes in podocyte structure and function in diabetes mellitus may lead to novel diagnostic tools and treatment strategies for diabetic nephropathy.

Alpha-hydroxy amides as a novel class of bradykinin B1 selective antagonists.

Antagonism of the bradykinin B(1) receptor represents a potential treatment for chronic pain and inflammation. Novel antagonists incorporating alpha-hydroxy amides were designed that display low-nanomolar affinity for the human bradykinin B(1) receptor and good bioavailability in the rat and dog. In addition, these functionally active compounds show high passive permeability and low susceptibility to phosphoglycoprotein mediated efflux, predictive of good CNS exposure.

Bradykinin B1 antagonists: biphenyl SAR studies in the cyclopropanecarboxamide series.

SAR study of the biphenyl region of cyclopropanecarboxamide derived bradykinin B(1) antagonists was examined. Incorporation of a pyridine in place of the proximal phenyl ring and chlorination of the distal phenyl ring proved to be well tolerated and provided compounds with improved pharmacokinetic profiles, CNS penetration, and enhanced receptor occupancy.

Identification of the glomerular podocyte as a target for growth hormone action.

GH excess in both the human and transgenic animal models is characterized by significant changes in blood pressure and renal function. The GH/GH receptor (GHR) axis is also implicated in the development of diabetic nephropathy. However, it is not clear whether GH's actions on renal function are due to indirect actions mediated via changes in blood pressure and vascular tone or due to direct action of GH on the kidney. We hypothesized that functional GHRs are expressed on the glomerular podocyte enabling direct actions of GH on glomerular function. Real-time PCR, immunohistochemistry, and Western blot analysis of murine podocyte cells (MPC-5) and kidney glomeruli demonstrated expression of GHR mRNA and protein. Exposure of both murine and human podocytes to GH (50-500 ng/ml) resulted in an increase in abundance of phosphorylated signal transducer and activator of transcription-5, Janus kinase-2, and ERK1/2 proteins. Exposure of podocytes to GH also caused changes in the intracellular distribution of the Janus kinase-2 adapter protein Src homology 2-Bbeta, stimulation of focal adhesion kinase, increase in reactive oxygen species, and GH-dependent changes in the actin cytoskeleton. We conclude that glomerular podocytes express functional GHRs and that GH increases levels of reactive oxygen species and induces reorganization of the actin cytoskeleton in these cells. These results provide a novel mechanistic link between GH's actions and glomerular dysfunction in disorders such as acromegaly and diabetic glomerulosclerosis.

Development of orally bioavailable and CNS penetrant biphenylaminocyclopropane carboxamide bradykinin B1 receptor antagonists.

A series of biphenylaminocyclopropane carboxamide based bradykinin B1 receptor antagonists has been developed that possesses good pharmacokinetic properties and is CNS penetrant. Discovery that the replacement of the trifluoropropionamide in the lead structure with polyhaloacetamides, particularly a trifluoroacetamide, significantly reduced P-glycoprotein mediated efflux for the series proved essential. One of these novel bradykinin B1 antagonists (13b) also exhibited suitable pharmacokinetic properties and efficient ex vivo receptor occupancy for further development as a novel approach for the treatment of pain and inflammation.

Amphetamine-induced locomotor activity is reduced in mice following MPTP treatment but not following selegiline/MPTP treatment.

MPTP treatment has been used in mice to cause dopaminergic neuronal cell loss and subsequent behavioral abnormalities. As such, this animal model is often used as a method for the characterization of putative novel therapeutics for disease states characterized by dopamine loss, such as Parkinson's disease. Previous reports of behavioral abnormalities in mice following MPTP intoxication, however, have been conflicting. For example, open field spontaneous activity has been reported to increase, decrease or not change in MPTP treated mice. Accordingly, a more robust and direct functional measure of MPTP-induced central dopamine depletion is needed. In the present manuscript, we report on the characterization of amphetamine-induced locomotor activity as a sensitive functional endpoint for dopamine loss following MPTP treatment. We found that the amphetamine-induced locomotor activity of C57BL/6 mice was reduced in a dose-dependent manner following treatment with MPTP. This reduction of activity was associated with decreases in central dopamine levels. Further, the potential for use of this endpoint to evaluate putative therapeutics is exemplified by the amelioration of these effects following pre-treatment with the MAO-B inhibitor selegiline.

5-Piperazinyl pyridine carboxamide bradykinin B1 antagonists.

A series of 2,3-diaminopyridine bradykinin B(1) antagonists was modified to mitigate the potential for bioactivation. Removal of the 3-amino group and incorporation of basic 5-piperazinyl carboxamides at the pyridine 5-position provided compounds with high affinity for the human B(1) receptor.

Cyclopropylamino acid amide as a pharmacophoric replacement for 2,3-diaminopyridine. Application to the design of novel bradykinin B1 receptor antagonists.

Antagonism of the bradykinin B1 receptor represents a potential treatment for chronic pain and inflammation. Novel antagonists were designed that display low-nanomolar affinity for the human bradykinin B1 receptor and good bioavailability in the rat.