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1.  Biological Evaluation of Benzothiazole Ethyl Urea Inhibitors of Bacterial Type II Topoisomerases 
Antimicrobial Agents and Chemotherapy  2013;57(12):5977-5986.
The type II topoisomerases DNA gyrase (GyrA/GyrB) and topoisomerase IV (ParC/ParE) are well-validated targets for antibacterial drug discovery. Because of their structural and functional homology, these enzymes are amenable to dual targeting by a single ligand. In this study, two novel benzothiazole ethyl urea-based small molecules, designated compound A and compound B, were evaluated for their biochemical, antibacterial, and pharmacokinetic properties. The two compounds inhibited the ATPase activity of GyrB and ParE with 50% inhibitory concentrations of <0.1 μg/ml. Prevention of DNA supercoiling by DNA gyrase was also observed. Both compounds potently inhibited the growth of a range of bacterial organisms, including staphylococci, streptococci, enterococci, Clostridium difficile, and selected Gram-negative respiratory pathogens. MIC90s against clinical isolates ranged from 0.015 μg/ml for Streptococcus pneumoniae to 0.25 μg/ml for Staphylococcus aureus. No cross-resistance with common drug resistance phenotypes was observed. In addition, no synergistic or antagonistic interactions between compound A or compound B and other antibiotics, including the topoisomerase inhibitors novobiocin and levofloxacin, were detected in checkerboard experiments. The frequencies of spontaneous resistance for S. aureus were <2.3 × 10−10 with compound A and <5.8 × 10−11 with compound B at concentrations equivalent to 8× the MICs. These values indicate a multitargeting mechanism of action. The pharmacokinetic properties of both compounds were profiled in rats. Following intravenous administration, compound B showed approximately 3-fold improvement over compound A in terms of both clearance and the area under the concentration-time curve. The measured oral bioavailability of compound B was 47.7%.
doi:10.1128/AAC.00719-13
PMCID: PMC3837865  PMID: 24041906
2.  Imidazo[1,2-a]pyridines That Directly Interact with Hepatitis C NS4B: Initial Preclinical Characterization 
ACS Medicinal Chemistry Letters  2012;3(7):565-569.
A series of imidazo[1,2-a]pyridines which directly bind to HCV Non-Structural Protein 4B (NS4B) is described. This series demonstrates potent in vitro inhibition of HCV replication (EC50 < 10 nM), direct binding to purified NS4B protein (IC50 < 20 nM), and an HCV resistance pattern associated with NS4B (H94N/R, V105L/M, F98L) that are unique among reported HCV clinical assets, suggestive of the potential for additive or synergistic combination with other small molecule inhibitors of HCV replication.
doi:10.1021/ml300090x
PMCID: PMC4025644  PMID: 24900511
NS4B; hepatitis C virus; imidazo[1,2-a]pyridines; replicon
3.  MATCH: An Atom- Typing Toolset for Molecular Mechanics Force Fields 
We introduce a toolset of program libraries collectively titled MATCH (Multipurpose Atom-Typer for CHARMM) for the automated assignment of atom types and force field parameters for molecular mechanics simulation of organic molecules. The toolset includes utilities for the conversion from multiple chemical structure file formats into a molecular graph. A general chemical pattern-matching engine using this graph has been implemented whereby assignment of molecular mechanics atom types, charges and force field parameters is achieved by comparison against a customizable list of chemical fragments. While initially designed to complement the CHARMM simulation package and force fields by generating the necessary input topology and atom-type data files, MATCH can be expanded to any force field and program, and has core functionality that makes it extendable to other applications such as fragment-based property prediction. In the present work, we demonstrate the accurate construction of atomic parameters of molecules within each force field included in CHARMM36 through exhaustive cross validation studies illustrating that bond increment rules derived from one force field can be transferred to another. In addition, using leave-one-out substitution it is shown that it is also possible to substitute missing intra and intermolecular parameters with ones included in a force field to complete the parameterization of novel molecules. Finally, to demonstrate the robustness of MATCH and the coverage of chemical space offered by the recent CHARMM CGENFF force field (Vanommeslaeghe, et al., JCC., 2010, 31, 671–690), one million molecules from the PubChem database of small molecules are typed, parameterized and minimized.
doi:10.1002/jcc.21963
PMCID: PMC3228871  PMID: 22042689
Force Fields; Atom Typing; Molecular Graph; Partial Charges; Parameterization
4.  Long-term Functional and Oncologic Results of Transoral Robotic Surgery for Oropharyngeal Squamous Cell Carcinoma 
Mayo Clinic Proceedings  2012;87(3):219-225.
Objective
To examine the long-term functional and oncologic results in patients who underwent transoral robotic surgery (TORS) as primary therapy or as part of combined therapy for oropharyngeal squamous cell carcinoma arising in the tonsil or base of tongue.
Patients and Methods
We reviewed a prospective TORS database of patients with squamous cell carcinoma arising in the tonsil or base of tongue treated between March 2007 and February 2009 to determine oncologic outcomes at 24 months or more of follow-up. The presenting tumor stage, histopathologic factors, surgical margins, and adjuvant treatment extent were evaluated. Functional outcomes included gastrostomy tube dependence and tracheostomy dependence. Oncologic outcomes included local, regional, and distant control and disease-specific and recurrence-free survival.
Results
A total of 66 TORS patients were followed up for a minimum of 2 years. Most (97.0%; 64 of 66) were able to eat orally within 3 weeks after surgery before starting adjuvant therapy. Long-term gastrostomy tube use was required in 3 of the 66 (4.5%) and long-term tracheotomy in 1 (1.5%). Three-year estimated local control and regional control were 97.0% and 94.0%, respectively. Two-year disease-specific survival and recurrence-free survival were 95.1% and 92.4%, respectively.
Conclusion
With appropriate adjuvant therapy, TORS achieves excellent functional results for patients with oropharyngeal squamous cell carcinoma. Oncologic outcomes are equivalent or superior to results of other surgical and nonsurgical treatments.
doi:10.1016/j.mayocp.2011.10.007
PMCID: PMC3538408  PMID: 22386176
5.  Strong asymmetric hydrogen bonding in 2-(oxamoylamino)ethyl­ammonium oxamate–oxamic acid (1/1) 
The title compound, C4H10N3O2 +·C2H2NO3 −·C2H3NO3, con­tains at least 11 distinct hydrogen-bond inter­actions showing a great variety of bond strengths. The shortest and strongest hydrogen bond [O⋯O = 2.5004 (12) Å] is found between the uncharged oxamic acid molecule and the oxamate mono­anion. The grouping formed by such a strong hydrogen bond can thus be considered as a hydrogen bis­(oxamate) monoanion. It lacks crystallographic symmetry and the two oxamate groups have different conformations, showing an asymmetric hydrogen-bond inter­action. Significantly, the asymmetry allows us to draw a direct comparison of site basicity for the two inequivalent carboxyl­ate O atoms in the planar oxamate anion. The constituent mol­ecular ions of (I) form ribbons, where all amide and carboxyl­ate groups are coplanar. Graph-set analysis of the hydrogen-bonded net­works reveals the R 2 2(10) and R 2 2(9) homodromic nets as important structure-directing motifs, which appear to be a common feature of many oxamate-containing compounds.
doi:10.1107/S0108270110004233
PMCID: PMC2855574  PMID: 20203413
6.  A new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) affects Soybean Asian rust (Phakopsora pachyrhizi) spore germination 
BMC Biotechnology  2011;11:14.
Background
Asian rust (Phakopsora pachyrhizi) is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP) leaves was cloned into the pGAPZα-B vector for expression in Pichia pastoris.
Results
A cDNA encoding a chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP), was isolated from leaves. The amino acid sequence predicts a (β/α)8 topology common to Class III Chitinases (glycoside hydrolase family 18 proteins; GH18), and shares similarity with other GH18 members, although it lacks the glutamic acid residue essential for catalysis, which is replaced by glutamine. CaclXIP was expressed as a recombinant protein in Pichia pastoris. Enzymatic assay showed that purified recombinant CaclXIP had only residual chitinolytic activity. However, it inhibited xylanases from Acrophialophora nainiana by approx. 60% when present at 12:1 (w/w) enzyme:inhibitor ratio. Additionally, CaclXIP at 1.5 μg/μL inhibited the germination of spores of Phakopsora pachyrhizi by 45%.
Conclusions
Our data suggests that CaclXIP belongs to a class of naturally inactive chitinases that have evolved to act in plant cell defence as xylanase inhibitors. Its role on inhibiting germination of fungal spores makes it an eligible candidate gene for the control of Asian rust.
doi:10.1186/1472-6750-11-14
PMCID: PMC3045311  PMID: 21299880
7.  Oncolysis by an HSV-1 engineered to express cytosine deaminase and a fusogenic glycoprotein for head and neck squamous cell carcinoma 
Background
A replication-competent, attenuated, oncolytic herpes simplex virus-1, OncoVEXGALV/CD, has previously been engineered to express a fusogenic protein from the gibbon ape leukemia virus and cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT) which converts fluorocytosine (5-FC) to 5-fluorouracil (5-FU). OncoVEXGFP is an analogous vector that expresses enhanced green fluorescent protein.
Methods
We assessed the ability of OncoVEXGALV/CD and OncoVEXGFP to infect, replicate within, and lyse four head and neck squamous carcinoma (HNSCC) cell lines in vitro. The effects of adding 5-FC with OncoVEXGALV/CD were evaluated.
Results
HNSCC was permissive to GFP expression in100% of cells by OncoVEXGFP at a multiplicity of infection (MOI) of 1 after 48 hours, and supported logarithmic viral replication. Virus caused >60% cell death six days after exposure to virus at MOI 0.1 in three of the four cell lines. 5-FC failed to enhance cytotoxicity induced by OncoVEXGALV/CD at MOI 0.1. However, for the least sensitive SCC25 cell line, virus at MOI 0.01 was cytotoxic to only 4% of cells after six days, but was cytotoxic to 35% of cells with 5-FC.
Conclusions
OncoVEXGALV/CD efficiently infects, replicates within and lyses HNSCC at relatively low viral doses. Prodrug conversion by CD did not enhance therapy at viral doses which cause efficient cytotoxicity, but may have beneficial effects in less sensitive cell lines at low viral doses.
doi:10.1001/archoto.2009.214
PMCID: PMC2824889  PMID: 20157061
9.  Synergy of a Herpes OncolyticVirus and Paclitaxel for Anaplastic Thyroid Cancer 
Purpose
Novel therapeutic regimens are needed to improve the dismal outcomes of patients with anaplastic thyroid cancer (ATC). Oncolytic herpes simplex virus have shown promising activity against human ATC. We studied the application of oncolytic herpes simplex virus (G207 and NV1023) in combination with currently used chemotherapeutic drugs (paclitaxel and doxorubicin) for the treatment of ATC.
Experimental Design and Results
All four agents showed dose-response cytotoxicity in vitro for the human ATC cell lines KAT4 and DRO90-1. G207, combined with paclitaxel, showed synergistic cytotoxicity. Chou-Talalay combination indices ranged from 0.56 to 0.66 for KAT4, and 0.68 to 0.74 for DRO90-1at higher affected fractions. Paclitaxel did not enhance G207 viral entry and early gene expression or G207 viral replication. Paclitaxel combined with G207 compared with single-agent treatment or controls showed significantly increased microtubule acetylation, mitotic arrest, aberrant chromatid separation, inhibition of metaphase to anaphase progression, and apoptosis. A single i.t. injection of G207 combined with biweekly i.p. paclitaxel injections in athymic nude mice bearing KAT4 flank tumors showed significantly reduced mean tumor volume (74 F 38 mm3) compared with G207 alone (388 F 109 mm3), paclitaxel alone (439 F 137 mm3), and control (520 F 160 mm3) groups at 16 days. There was no morbidity in vivo attributable to therapy.
Conclusions
Mechanisms of paclitaxel antitumoral activity, including microtubule acetylation, mitotic block, and apoptosis, were enhanced by G207, which also has direct oncolytic effects. Combination of G207 and paclitaxel therapy is synergistic in treating ATC and holds promise for patients with this fatal disease.
doi:10.1158/1078-0432.CCR-07-4628
PMCID: PMC2719715  PMID: 18316577
10.  Introduction of a portable ultrasound unit into the health services of the Lugufu refugee camp, Kigoma District, Tanzania 
Background
Diagnostic imaging services are scarce in much of the developing world. Ultrasound is a low-cost, safe, and widely applicable imaging modality.
Aims
We delivered a portable ultrasound machine to the Lugufu refugee camp in Tanzania and conducted a course on its use in order to assess the feasibility of introducing this technology into a very low-resource setting.
Methods
We conducted an intensive 4-day ultrasound training course for health care providers at Lugufu and established an exam logbook to track the frequency of use, distribution of ultrasound applications, and gender and age distribution of patients.
Results
Between 4 October 2005 and 1 November 2007 547 separate ultrasound exams on 460 patients were recorded at Lugufu. Overall, 86% of patients studied were female. Pregnancy-related exams accounted for 24.1% of total usage. The 20- to 29-year-old age group accounted for over one third of total exams performed.
Conclusion
Ultrasound is a feasible and sustainable imaging modality in a very low-resource setting such as the Lugufu refugee camp in Tanzania.
doi:10.1007/s12245-008-0074-7
PMCID: PMC2657264  PMID: 19384640
Ultrasound; Refugee camp; Developing world; Imaging
11.  Coiled-Coils at the Edge of Configurational Heterogeneity. Structural Analyses of Parallel and Antiparallel Homotetrameric Coiled-Coils Reveal Configurational Sensitivity to a Single Solvent-Exposed Amino Acid Substitution.†§ 
Biochemistry  2006;45(14):4463-4473.
A detailed understanding of the mechanisms by which particular amino acid sequences can give rise to more than one folded structure, such as for proteins that undergo large conformational changes or misfolding, is a long-standing objective of protein chemistry. Here we describe the crystal structures of a single coiled-coil peptide in distinct parallel and antiparallel tetrameric configurations and further describe the parallel or antiparallel crystal structures of several related peptide sequences; the antiparallel tetrameric assemblies represents the first crystal structures of GCN4-derived peptides exhibiting such a configuration. Intriguingly, substitution of a single solvent-exposed residue enabled the parallel coiled-coil tetramer GCN4-pLI to populate the antiparallel configuration, suggesting that the two configurations are close enough in energy for subtle sequence changes to have important structural consequences. We present a structural analysis of the small changes to helix register and side chain conformations that accommodate the two configurations, and have supplemented these results using solution studies and a molecular dynamics energetic analysis using a replica exchange methodology. Considering the previous examples of structural nonspecificity in coiled-coil peptides, the findings reported here not only emphasize the predisposition of the coiled-coil motif to adopt multiple configurations, but also call attention to the associated risk that observed crytstal structures may not represent the only (or even the major) species present in solution.
doi:10.1021/bi060092q
PMCID: PMC1780269  PMID: 16584182

Results 1-12 (12)