Search

Structure-guided drug design led to new alkylamine renin inhibitors with improved in vitro and in vivo potency. Lead compound 21a, has an IC(50) of 0.83nM for the inhibition of human renin in plasma (PRA). Oral administration of 21a at 10mg/kg resulted in >20h reduction of blood pressure in a double transgenic rat model of hypertension., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Structure-based drug design led to the identification of a novel class of potent, low MW alkylamine renin inhibitors. Oral administration of lead compound 21l, with MW of 508 and IC(50) of 0.47nM, caused a sustained reduction in mean arterial blood pressure in a double transgenic rat model of hypertension.

Background: The renin-angiotensin-aldosterone system (RAS) cascade is a major target for the clinical management of hypertension. Although inhibitors of various components of this cascade have been developed successfully, development of renin inhibitors has proven to be problematic. The development of these inhibitors has been hindered by poor bioavailability and complex synthesis. However, despite the challenges of designing renin inhibitors, the enzyme remains a promising target for the development of novel treatments for hypertension. X-ray crystallographic data could greatly assist the design and development of these inhibitors. Here we describe the purification and characterization of recombinant human renin for x-ray crystallization studies., Results: A cDNA encoding the full length of native human preprorenin (406 amino acid residues) was introduced into the HEK-293 cell line. A clonal cell line expressing prorenin was generated and grown under serum free conditions in a hollow fiber bioreactor. Prorenin was constitutively secreted and purified directly from the conditioned medium. Concanavalin A chromatography effectively enriched and purified prorenin to 90% homogeneity in a single step. Prorenin was converted to active renin by trypsin digestion to remove the propeptide. Active renin was further purified using a cation exchange column followed by a gel filtration column. Biochemical characterization of the recombinant enzyme showed both binding and catalytic properties were essentially identical to previously reported activities for purified renin. Crystals were grown using this material in our X-ray structure studies, and high resolution diffraction was obtained., Conclusion: This present work describes a simple and efficient method for the generation and purification of active human renin. The protein is highly pure and is suitable for supporting structural biology efforts.

A cDNA encoding human prepro-cathepsin E was introduced into the adenovirus-transformed HEK-293 (human embryonic kidney) cell line. The construct contained both a V5 peptide epitope and histidine tags at the carboxy terminus. Transfected cells efficiently secreted recombinant pro-cathepsin E into the culture medium. The secreted pro-cathepsin E was purified in a single step using Ni affinity chromatography yielding a protein of about 92 kDa under non-reducing conditions. The amino-terminal sequence of the purified protein began at Ser20, suggesting human cathepsin E accumulated in the culture supernatant as the pro-enzyme. The purified protein was rapidly and completely converted to the active form by treatment at pH 4.0 or below. Steady state kinetic parameters for hydrolysis of the fluorogenic peptide substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-d-Arg-NH2 (cleavage at the Phe-Phe bond) were consistent with previously reported values for purified human enzyme (kc/Ki= 53 x 10(6) M(-1) s(-1), Km= 6.3 microM, and kcat= 3 x 10(2) s(-1)). The activated protein was potently inhibited by pepstatin with Ki= 0.2 nM, as well as a reported beta secretase inhibitor. This work demonstrates the potential for producing large quantities of highly purified human cathepsin E from HEK-293 cells in quantities to support both biochemical and structural characterization of the enzyme.

As the global threat of drug- and antibiotic-resistant bacteria continues to rise, new strategies are required to advance the drug discovery process. This work describes the construction of an array of Escherichia coli strains for use in whole-cell screens to identify new antimicrobial compounds. We used the recombination systems from bacteriophages lambda and P1 to engineer each strain in the array for low-level expression of a single, essential gene product, thus making each strain hypersusceptible to specific inhibitors of that gene target. Screening of nine strains from the array in parallel against a large chemical library permitted identification of new inhibitors of bacterial growth. As an example of the target specificity of the approach, compounds identified in the whole-cell screen for MurA inhibitors were also found to block the biochemical function of the target when tested in vitro.

The physical map of the 2.1 megabase chromosome of Streptococcus mutans GS-5 has been refined by including all ApaI and SmaI fragments of 5 kbp or greater, and by positioning the fragments generated by the endonuclease I-CeuI. Sixty-three new genetic loci have been added to the map, so that it now contains 90 loci. The new loci include those for 35 cloned streptococcal genes of established function and for 23 S. mutans genes of putative function. In addition, five rrn operons were identified and placed on the map of the chromosome. The presence of a SmaI site in each of the rrn operons allowed the direction of transcription of each operon to be deduced. The orientation of the rrn loci indicates that their transcription is directed away from a small region of the chromosome, identifying a possible region for the initiation of chromosome replication.

In this study classical ribotyping based on hybridization of an enteroccocal ribosomal operon previously cloned from Enterococcus hirae (Sechi and Daneo-Moore, 1993) with XbaI cut chromosomal DNA and PCR-ribotyping were used to characterize the molecular epidemiology of 131 Enterococcus faecium, with high-level resistance to gentamicin, isolated from different hospitals in Italy and the United States. The ribotyping was able to differentiate all 131 clinical isolates into 96 family patterns. These family patterns appeared to be useful in establishing epidemiological spread. The results obtained were in agreement with those previously published, suggesting the presence of five to six operons in the Enterococcus genus (Sechi et al., 1994). We performed PCR-ribotyping, based on conserved sequences at the 3' end of the enterococcal 16S rrn and the 5' end of the 23S rrn, on 131 clinical isolates as well as on several enterococcal ATCC strains tested. The results were then compared with those obtained with the classical ribotyping method. The results suggest the presence of at least four classes of intergenic spacers among enterococci, but these classes are not helpful in differentiating between Enterococci or among Enterococcal isolates.