A Podocyte-Based Automated Screening Assay Identifies Protective Small Molecules.

Podocyte injury and loss mark an early step in the pathogenesis of various glomerular diseases, making these cells excellent targets for therapeutics. However, cell-based high-throughput screening assays for the rational development of podocyte-directed therapeutics are currently lacking. Here, we describe a novel high-content screening-based phenotypic assay that analyzes thousands of podocytes per assay condition in 96-well plates to quantitatively measure dose-dependent changes in multiple cellular features. Our assay consistently produced a Z' value >0.44, making it suitable for compound screening. On screening with >2100 pharmacologically active agents, we identified 24 small molecules that protected podocytes against injury in vitro (1% hit rate). Among the identified hits, we confirmed an ß1-integrin agonist, pyrintegrin, as a podocyte-protective agent. Treatment with pyrintegrin prevented damage-induced decreases in F-actin stress fibers, focal adhesions, and active ß1-integrin levels in cultured cells. In vivo, administration of pyrintegrin protected mice from LPS-induced podocyte foot process effacement and proteinuria. Analysis of the murine glomeruli showed that LPS administration reduced the levels of active ß1 integrin in the podocytes, which was prevented by cotreatment with pyrintegrin. In rats, pyrintegrin reduced peak proteinuria caused by puromycin aminonucleoside-induced nephropathy. Our findings identify pyrintegrin as a potential therapeutic candidate and show the use of podocyte-based screening assays for identifying novel therapeutics for proteinuric kidney diseases.

Supramolecular assemblies of novel aminonucleoside phospholipids and their bonding to nucleic acids.

A novel class of aminonucleoside phospholipids has been developed. These molecules could spontaneously assemble into supramolecular structures including multilamellar organization, hydrogels, superhelical strands, and vesicles. Their ability to bind to DNA by hydrogen bonding and π-π stacking interactions was investigated by many means.

ß-Puromycin selection of modified ribosomes for in vitro incorporation of ß-amino acids.

Ribosomally mediated protein biosynthesis is limited to α-L-amino acids. A strong bias against ß-L-amino acids precludes their incorporation into proteins in vivo and also in vitro in the presence of misacylated ß-aminoacyl-tRNAs. Nonetheless, earlier studies provide some evidence that analogues of aminoacyl-tRNAs bearing ß-amino acids can be accommodated in the ribosomal A-site. Both functional and X-ray crystallographic data make it clear that the exclusion of ß-L-amino acids as participants in protein synthesis is a consequence of the architecture of the ribosomal peptidyltransferase center (PTC). To enable the reorganization of ribosomal PTC architecture through mutagenesis of 23S rRNA, a library of modified ribosomes having modifications in two regions of the 23S rRNA (2057-2063 and 2496-2507 or 2582-2588) was prepared. A dual selection procedure was used to obtain a set of modified ribosomes able to carry out protein synthesis in the presence ß-L-amino acids and to provide evidence for the utilization of such amino acids, in addition to α-L-amino acids. ß-Puromycin, a putative mimetic for ß-aminoacyl-tRNAs, was used to select modified ribosome variants having altered PTC architectures, thus potentially enabling incorporation of ß-L-amino acids. Eight types of modified ribosomes altered within the PTC have been selected by monitoring improved sensitivity to ß-puromycin in vivo. Two of the modified ribosomes, having 2057AGCGUGA2063 and 2502UGGCAG2507 or 2502AGCCAG2507, were able to suppress UAG codons in E. coli dihydrofolate reductase (DHFR) and scorpion Opisthorcanthus madagascariensis peptide IsCT mRNAs in the presence of ß-alanyl-tRNA(CUA).

Structural investigations of mode of action of drugs: structure and conformation of a novel peptidyl nucleoside antibiotic chryscandin hydrochloride pentahydrate.

The structure and conformation of antibiotic chryscandin, a structural analog of the terminus 3'-aminoacyladenylate moiety of tRNA, has been determined. The crystals belong to orthorhombic space group P2(1)2(1)2(1). The structure was refined to an R value of 0.065 for 1872 reflections. The structure and conformation has been compared with that of puromycin. The sugar pucker is different from that of puromycin, thus creating different orientation of peptide group with respect to nucleoside. All the water molecules are involved in hydrogen bonding. The crystal is also stabilized by stacking of adenine bases and p-methoxyphenyl rings. The results will be helpful in understanding structure-biological activity relationships, identification of inhibitors of metallopeptidases, and how chryscandin interacts with ribosomal units.

Comparative studies of coordination properties of puromycin and puromycin aminonucleoside towards copper(II) ions.

Protonation equilibria of puromycin (PM) and puromycin aminonucleoside (PAN) and their coordination by copper(II) ion were studied in solution by potentiometry, electronic absorption spectroscopy (UV-Vis), circular dichroism (CD), electron paramagnetic resonance (EPR) and mass spectrometry. For puromycin four mononuclear complexes were found, with stoichiometries Cu(PM)2+, CuH(-1)(PM)+, CuH(-2)(PM) and CuH(-3)(PM)(-). In each of them the Cu(II) ion was bound in the peptidic-like manner, the differences of stoichiometries are a consequence of subsequent deprotonations of the sugar C2'-OH group and the coordinated water molecule. The coordination mode for puromycin aminonucleoside was aminosugar-like. Two dimeric complexes, Cu2H(-1)(PAN)2(2+) and Cu2H(-2)(PAN)2+, and one monomeric CuH(-2)(PAN)2 were found. The N6,N6-dimethyladenine moiety of PAN was not involved in the coordination process due to steric hindrance.

The pur6 gene of the puromycin biosynthetic gene cluster from Streptomyces alboniger encodes a tyrosinyl-aminonucleoside synthetase.

The pur6 gene of the puromycin biosynthetic gene (pur) cluster from Streptomyces alboniger is shown to be essential for puromycin biosynthesis. Cell lysates from this mycelial bacterium were active in linking L-tyrosine to both 3'-amino-3'-deoxyadenosine and N6,N6-dimethyl-3'-amino-3'-deoxyadenosine with a peptide-like bond. Identical reactions were performed by cell lysates from Streptomyces lividans or Escherichia coli transformants that expressed pur6 from a variety of plasmid constructs. Physicochemical and biochemical analyses suggested that their products were tridemethyl puromycin and O-demethylpuromycin, respectively. Therefore, it appears that Pur6 is the tyrosinyl-aminonucleoside synthetase of the puromycin biosynthetic pathway.

Cystocin, a novel antibiotic, produced by Streptomyces sp. GCA0001: biological activities.

Cystocin belongs to the class of nucleoside antibiotics from Streptomyces sp. GCA0001. Cystocin showed good activity against gram-positive bacteria, but showed less activity against the gram-negative bacteria. Cystocin exhibited about two to four folds higher activity than puromycin. Especially, cystocin shows relatively strong activity against Streptococcus strains. Cystocin shows quite potent antitumor activity against all of the cells tested showing IC50 values of 0.10 to 0.14 microg/mL. This in vitro result indicates that the cytotoxocity of cystocin is two ten folds more active than puromycin's.

Identification of a set of genes involved in the biosynthesis of the aminonucleoside moiety of antibiotic A201A from Streptomyces capreolus.

A novel cosmid (pABC6.5) whose DNA insert from Streptomyces capreolus, the A201A antibiotic producer, overlaps the inserts of the previously reported pCAR11 and pCAR13 cosmids, has been isolated. These two latter cosmids were known to contain the aminonucleoside antibiotic A201A resistance determinants ard2 and ard1, respectively. Together, these three cosmids have permitted the identification of a DNA stretch of 19 kb between ard1 and ard2, which should comprise a large region of a putative A201A biosynthetic (ata) gene cluster. The sequence of the 7 kb upstream of ard1 towards ard2 reveals seven consecutive open reading frames: ataP3, ataP5, ataP4, ataP10, ataP7, ata12 and ataPKS1. Except for the last two, their deduced products present high similarities to an identical number of counterparts from the pur cluster of Streptomyces alboniger that were either known or proposed to be implicated in the biosynthesis of the N6,N6-dimethyl-3'-amino-3'-deoxyadenosine moiety of puromycin. Because A201A contains this chemical moiety, these ataP genes are most likely implicated in its biosynthesis. Accordingly, the ataP4, ataP5 and ataP10 genes complemented specific puromycin nonproducing Deltapur4, Deltapur5 and Deltapur10 mutants of S. alboniger, respectively. Amino acid sequence comparisons suggest that ata12 and ataPKS1 could be implicated in the biosynthesis of the d-rhamnose and alpha-p-coumaric acid moieties of A201A. Further sequencing of 2 kb of DNA downstream of ard1 has disclosed a region which might contain one end of the ata cluster.

Substituted purines elicit differential cytokinetic, molecular and phenotypic effects in HL-60 cells.

This study investigated and compared the cytokinetic, phenotypic and molecular effects elicited in HL-60 human leukemic cells by low doses (0.6 microM) of 3 closely related, substituted purines, puromycin (PM), puromycin aminonucleoside (PAN) and 6-dimethylaminopurine (6-DMAP). PM, but not 6-DMAP or PAN, inhibited [14C]leucine incorporation, induced a time-related cytotoxic effect, a G2-arrest, a metaphase-mitotic-arrest, apoptosis and c-myc mRNA superinduction. PAN and 6-DMAP exerted no detectable morphological or cytokinetic effects, although exposure to these drugs resulted in downregulation of c-myc mRNA levels. We suggest that the specific effects exerted by PM relate to the generation of nascent peptidyl-PM complexes by PM, but not by 6-DMAP or PAN.