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Ribble W.,Replidyne | Hill W.E.,University of Montana | Ochsner U.A.,Replidyne | Ochsner U.A.,Crestone, Inc. | And 8 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2010

Bacterial protein synthesis is the target for numerous natural and synthetic antibacterial agents. We have developed a poly(U) mRNA-directed aminoacylation/translation protein synthesis system composed of phenyltRNA synthetases, ribosomes, and ribosomal factors from Escherichia coli. This system, utilizing purified components, has been used for high-throughput screening of a small-molecule chemical library. We have identified a series of compounds that inhibit protein synthesis with 50% inhibitory concentrations (IC 50s) ranging from 3 to 14 μM. This series of compounds all contained the same central scaffold composed of tetrahydropyrido[4,3-d] pyrimidin-4-ol (e.g., 4H-pyridopyrimidine). All analogs contained an ortho pyridine ring attached to the central scaffold in the 2 position and either a five- or a six-member ring tethered to the 6-methylene nitrogen atom of the central scaffold. These compounds inhibited the growth of E. coli, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, with MICs ranging from 0.25 to 32 μg/ml. Macromolecular synthesis (MMS) assays with E. coli and S. aureus confirmed that antibacterial activity resulted from specific inhibition of protein synthesis. Assays were developed for the steps performed by each component of the system in order to ascertain the target of the compounds, and the ribosome was found to be the site of inhibition. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Gill S.C.,Replidyne | Rubino C.M.,Institute for Clinical Pharmacodynamics | Bassett J.,U.S. Army | Miller L.,U.S. Army | And 8 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2010

There are few options for prophylaxis after exposure to Bacillus anthracis, especially in children and women of childbearing potential. Faropenem is a β-lactam in the penem subclass that is being developed as an oral prodrug, faropenem medoxomil, for the treatment of respiratory tract infections. Faropenem was shown to have in vitro activity against B. anthracis strains that variably express the bla1 β-lactamase (MIC range, ≤0.06 to 1 μg/ml). In this study we evaluated the pharmacokinetic-pharmacodynamic (PK-PD) relationships between the plasma faropenem free-drug (f) concentrations and efficacy against B. anthracis in a murine postexposure prophylaxis inhalation model. The plasma PKs and PKs-PDs of faropenem were evaluated in BALB/c mice following the intraperitoneal (i.p.) administration of doses ranging from 2.5 to 160 mg/kg of body weight. For the evaluation of efficacy, mice received by inhalation aerosol doses of B. anthracis (Ames strain; faropenem MIC, 0.06 μg/ml) at 100 times the 50% lethal dose. The faropenem dosing regimens (10, 20, 40, and 80 mg/kg/day) were administered i.p. at 24 h postchallenge at 4-, 6-, and 12-h intervals for 14 days. The sigmoid maximum-threshold-of-efficacy (Emax) model fit the survival data, in which the free-drug area under the concentration-time curve (fAUC)/MIC ratio, the maximum concentration of free drug in plasma (fCmax)/MIC ratio, and the cumulative percentage of a 24-h period that the free-drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (f %TMIC) were each evaluated. Assessment of f %TMIC demonstrated the strongest correlation with survival (R2 = 0.967) compared to the correlations achieved by assessment of fAUC/MIC or fCmax/MIC, for which minimal correlations were observed. The 50% effective dose (ED50), ED 90, and ED99 corresponded to f %TMIC values of 10.6, 13.4, and 16.4%, respectively, and Emax was 89.3%. Overall, faropenem demonstrated a high level of activity against B. anthracis in the murine postexposure prophylaxis inhalation model. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Shaikh N.,University of Pittsburgh | Wang E.E.L.,Replidyne | Arguedas A.,University of Medical Sciences of Costa Rica | Dagan R.,Pediatric Infectious Disease Unit | And 3 more authors.
Pediatric Infectious Disease Journal | Year: 2011

We followed symptoms of children with acute otitis media (AOM), who were enrolled in a clinical trial that included a baseline tympanocentesis. We observed marked and rapid improvement in symptom scores after tympanocentesis. Although symptom scores (measured by the AOM-SOS) correlated with overall clinical assessment and bacteriologic outcome, the early effect of tympanocentesis rendered the AOM-SOS less useful as a primary outcome measure. © 2011 Lippincott Williams & Wilkins, Inc.


Ribble W.,Replidyne | Kane S.D.,Replidyne | Bullard J.M.,Replidyne | Bullard J.M.,University of Texas–Pan American
Enzyme Research | Year: 2015

DNA replication in bacteria is accomplished by a multicomponent replicase, the DNA polymerase III holoenzyme (pol III HE). The three essential components of the pol III HE are the α polymerase, the β sliding clamp processivity factor, and the DnaX clamp-loader complex. We report here the assembly of the functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme capable of DNA synthesis consists of α, β and DnaX (τ and γ), δ and δ ′ components of the clamp-loader complex. The proteins were each cloned and expressed in a native form. Each component of the system was purified extensively. The minimum holoenzyme from these five purified subunits reassembled is sufficient for rapid and processive DNA synthesis. In an isolated form the α polymerase was found to be unstable at temperatures above 65°C. We were able to increase the thermostability of the pol III HE to 98°C by addition and optimization of various buffers and cosolvents. In the optimized buffer system we show that a replicative polymerase apparatus, Tth pol III HE, is capable of rapid amplification of regions of DNA up to 15,000 base pairs in PCR reactions. © 2015 Wendy Ribble et al.


PubMed | University of Texas–Pan American and Replidyne
Type: | Journal: Enzyme research | Year: 2015

DNA replication in bacteria is accomplished by a multicomponent replicase, the DNA polymerase III holoenzyme (pol III HE). The three essential components of the pol III HE are the polymerase, the sliding clamp processivity factor, and the DnaX clamp-loader complex. We report here the assembly of the functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme capable of DNA synthesis consists of , and DnaX ( and ), and components of the clamp-loader complex. The proteins were each cloned and expressed in a native form. Each component of the system was purified extensively. The minimum holoenzyme from these five purified subunits reassembled is sufficient for rapid and processive DNA synthesis. In an isolated form the polymerase was found to be unstable at temperatures above 65C. We were able to increase the thermostability of the pol III HE to 98C by addition and optimization of various buffers and cosolvents. In the optimized buffer system we show that a replicative polymerase apparatus, Tth pol III HE, is capable of rapid amplification of regions of DNA up to 15,000 base pairs in PCR reactions.


Faqi A.S.,MPI Research | Lanphear C.,MPI Research | Gill S.,Replidyne | Colagiovanni D.B.,Replidyne
Reproductive Toxicology | Year: 2010

We determined the toxicity of faropenem medoxomil (FPM) in neonatal/juvenile dogs following 28 days of administration. The puppies received vehicle or FPM beginning on Postnatal Day (PND) 22 at respective dose levels of 0, 100, 300, 600, or 1400mg/kg-d (four daily doses (QID) of 25, 75, 150, or 350mg/kg/dose), respectively, at a dose volume of 5mL/kg/dose. Body weight, food consumption, clinical observation, clinical pathology, urine analysis and TK were evaluated. Body weight in males and kidney findings at 1400mg/kg-d were considered adverse. Comparison of Day 27 TK values with Day 1 parameters showed a change in FPM pharmacokinetic behavior over time with an apparent increase in the rate of clearance characterized by a decrease in AUC0-6 and Tmax values on Day 27 with little to no change in Cmax values. Based on these results, the No Observed Adverse Effect Level was 600mg/kg-d. © 2010 Elsevier Inc.


PubMed | Replidyne
Type: Journal Article | Journal: Organic and medicinal chemistry letters | Year: 2012

We have identified a series of compounds that inhibit protein synthesis in bacteria. Initial IC50s in aminoacylation/translation (A/T) assays ranged from 3 to14 M. This series of compounds are variations on a 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-ol scaffold (e.g., 4H-pyridopyrimidine).Greater than 80 analogs were prepared to investigate the structure-activity relationship (SAR). Structural modifications included changes in the central ring and substituent modifications in its periphery focusing on the 2- and 6-positions. An A/T system was used to determine IC50 values for activity of the analogs in biochemical assays. Minimum inhibitory concentrations (MIC) were determined for each analog against cultures of Enterococcus faecalis, Moraxella catarrhalis, Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli tolC mutants and E. coli modified with PMBN.Modifications to the 2-(pyridin-2-yl) ring resulted in complete inactivation of the compounds. However, certain modifications at the 6-position resulted in increased antimicrobial potency. The optimized compounds inhibited the growth of E. faecalis, M. catarrhalis, H. influenzae, S. pneumoniae, S. aureus, E. coli tolC, mutants and E. coli modified with PMBN with MIC values of 4, 0.12, 1, 2, 4, 1, 1 g/ml, respectively. IC50 values in biochemical assay were reduced to mid-nanomolar range.4H-pyridopyrimidine analogs demonstrate broad-spectrum inhibition of bacterial growth and modification of the compounds establishes SAR.


PubMed | Replidyne
Type: Journal Article | Journal: Antimicrobial agents and chemotherapy | Year: 2010

There are few options for prophylaxis after exposure to Bacillus anthracis, especially in children and women of childbearing potential. Faropenem is a beta-lactam in the penem subclass that is being developed as an oral prodrug, faropenem medoxomil, for the treatment of respiratory tract infections. Faropenem was shown to have in vitro activity against B. anthracis strains that variably express the bla1 beta-lactamase (MIC range,

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