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1.  Pyrrolidinobenzoic Acid Inhibitors of Influenza Virus Neuraminidase: the Hydrophobic Side Chain Influences Type A Subtype Selectivity 
Bioorganic & medicinal chemistry  2012;20(14):4582-4589.
Neuraminidase (NA) plays a critical role in the life cycle of influenza virus and is a target for new therapeutic agents. A series of influenza neuraminidase inhibitors with the pyrrolidinobenzoic acid scaffold containing lipophilic side chains at the C3 position have been synthesized and evaluated for influenza neuraminidase inhibitory activity. The size and geometry of the C3 side chains have been modified in order to investigate structure-activity relationships. The results indicated that size and geometry of the C3-side chain are important for selectivity of inhibition against N1 vs N2 NA, important type A influenza variants that infect man, including the highly lethal avian influenza.
doi:10.1016/j.bmc.2012.05.001
PMCID: PMC3401542  PMID: 22677529
Neuraminidase; avian influenza; pyrrolidinobenzoic acid
2.  Crystal structure of a new benzoic acid inhibitor of influenza neuraminidase bound with a new tilt induced by overpacking subsite C6 
Background
Influenza neuraminidase (NA) is an important target for antiviral inhibitors since its active site is highly conserved such that inhibitors can be cross-reactive against multiple types and subtypes of influenza. Here, we discuss the crystal structure of neuraminidase subtype N9 complexed with a new benzoic acid based inhibitor (2) that was designed to add contacts by overpacking one side of the active site pocket. Inhibitor 2 uses benzoic acid to mimic the pyranose ring, a bis-(hydroxymethyl)-substituted 2-pyrrolidinone ring in place of the N-acetyl group of the sialic acid, and a branched aliphatic structure to fill the sialic acid C6 subsite.
Results
Inhibitor 2 {4-[2,2-bis(hydroxymethyl)-5-oxo-pyrrolidin-1-yl]-3-[(dipropylamino)methyl)]benzoic acid} was soaked into crystals of neuraminidase of A/tern/Australia/G70c/75 (N9), and the structure refined with 1.55 Å X-ray data. The benzene ring of the inhibitor tilted 8.9° compared to the previous compound (1), and the number of contacts, including hydrogen bonds, increased. However, the IC50 for compound 2 remained in the low micromolar range, likely because one propyl group was disordered. In this high-resolution structure of NA isolated from virus grown in chicken eggs, we found electron density for additional sugar units on the N-linked glycans compared to previous neuraminidase structures. In particular, seven mannoses and two N-acetylglucosamines are visible in the glycan attached to Asn200. This long, branched high-mannose glycan makes significant contacts with the neighboring subunit.
Conclusions
We designed inhibitor 2 with an extended substituent at C4-corresponding to C6 of sialic acid-to increase the contact surface in the C6-subsite and to force the benzene ring to tilt to maximize these interactions while retaining the interactions of the carboxylate and the pyrolidinone substituents. The crystal structure at 1.55 Å showed that we partially succeeded in that the ring in 2 is tilted relative to 1 and the number of contacts increased, but one hydrophobic branch makes no contacts, perhaps explaining why the IC50 did not decrease. Future design efforts will include branches of unequal length so that both branches may be accommodated in the C6-subsite without conformational disorder. The high-mannose glycan attached to Asn200 makes several inter-subunit contacts and appears to stabilize the tetramer.
doi:10.1186/1472-6807-12-7
PMCID: PMC3416664  PMID: 22559154
Influenza neuraminidase inhibitor; Enzyme-ligand complex; Antiviral; Structure-based drug design; Glycoprotein; Glycan structure; Influenza virus; Benzoic acid; Pyrrolidinone
3.  SAR Studies for a New Class of Antibacterial NAD Biosynthesis Inhibitors 
A new lead class of antibacterial drug-like NAD synthetase (NADs) inhibitors was previously identified from a virtual screening study. Here a solution-phase synthetic library of 76 compounds, analogs of the urea-sulfonamide 5838, was synthesized in parallel to explore SAR on the sulfonamide aryl group. All library members were tested for enzyme inhibition against NADs and nicotinic acid mononucleotide adenylyltransferase (NaMNAT), the last two enzymes in the biosynthesis of NAD, and for growth inhibition in a B. anthracis antibacterial assay. Most compounds that inhibited bacterial growth also showed inhibition against one of the enzymes tested. While only modest enhancements in the enzyme inhibition potency against NADs were observed, of significance was the observation that the antibacterial urea-sulfonamides more consistently inhibited NaMNAT.
doi:10.1021/cc9000357
PMCID: PMC2888690  PMID: 19408950
4.  Virtual Screening to Identify Lead Inhibitors for Bacterial NAD Synthetase (NADs) 
Virtual screening was employed to identify new drug-like inhibitors of NAD synthetase (NADs) as antibacterial agents. Four databases of commercially available compounds were docked against three subsites of the NADs active site using FlexX in conjunction with CScore. Over 200 commercial compounds were purchased and evaluated in enzyme inhibition and antibacterial assays. 18 compounds inhibited NADs at or below 100 μM (7.6% hit rate), and two were selected for future SAR studies.
doi:10.1016/j.bmcl.2009.02.034
PMCID: PMC2666046  PMID: 19249205
5.  Prevention of KLF4-mediated tumor initiation and malignant transformation by UAB30 rexinoid 
Cancer biology & therapy  2009;8(3):289-298.
The transcription factor KLF4 acts in post-mitotic epithelial cells to promote differentiation, and functions in a context-dependent fashion as an oncogene. In the skin KLF4 is co-expressed with the nuclear receptors RARγ and RXRα, and formation of the skin permeability barrier is a shared function of these three proteins. We utilized a KLF4-transgenic mouse model of skin cancer in combination with cultured epithelial cells to examine functional interactions between KLF4 and retinoic acid receptors. In cultured cells, activation of a conditional, KLF4-estrogen receptor fusion protein by 4-hydroxytamoxifen resulted in rapid upregulation of transcripts for nuclear receptors including RARγ and RXRα. We tested retinoids in epithelial cell transformation assays, including an RAR-selective agonist (all-trans RA), an RXR-selective agonist (9-cis UAB30, rexinoid), and a pan agonist (9-cis RA). Unlike for several other genes, transformation by KLF4 was inhibited by each retinoid, implicating distinct nuclear receptor heterodimers as modulators of KLF4 transforming activity. When RXRα expression was suppressed by RNAi in cultured cells, transformation was promoted and the inhibitory effect of 9-cis UAB30 was attenuated. Similarly as shown for other mouse models of skin cancer, rexinoid prevented skin tumor initiation resulting from induction of KLF4 in basal keratinocytes. Rexinoid permitted KLF4 expression and KLF4-induced cell cycling, but attenuated the KLF4-induced misexpression of cytokeratin 1 in basal cells. Neoplastic lesions including hyperplasia, dysplasia and squamous cell carcinoma-like lesions were prevented for up to 30 days. Taken together, the results identify retinoid receptors including RXRα as ligand-dependent inhibitors of KLF4-mediated transformation or tumorigenesis.
PMCID: PMC2776760  PMID: 19197145
KLF4; tumor initiation; squamous cell carcinoma; retinoid; rexinoid; nuclear receptors; chemoprevention
6.  Structure of nicotinic acid mononucleotide adenylyltransferase from Bacillus anthracis  
The crystal structure of apo nicotinic acid mononucleotide adenylyltransferase (NaMNAT) from B. anthracis was determined to 2.3 Å resolution and compared with other bacterial NaMNAT structures.
Nicotinic acid mononucleotide adenylyltransferase (NaMNAT; EC 2.7.7.18) is the penultimate enzyme in the biosynthesis of NAD+ and catalyzes the adenylation of nicotinic acid mononucleotide (NaMN) by ATP to form nicotinic acid adenine dinucleotide (NaAD). This enzyme is regarded as a suitable candidate for antibacterial drug development; as such, Bacillus anthracis NaMNAT (BA NaMNAT) was heterologously expressed in Escherichia coli for the purpose of inhibitor discovery and crystallography. The crystal structure of BA NaMNAT was determined by molecular replacement, revealing two dimers per asymmetric unit, and was refined to an R factor and R free of 0.228 and 0.263, respectively, at 2.3 Å resolution. The structure is very similar to that of B. subtilis NaMNAT (BS NaMNAT), which is also a dimer, and another independently solved structure of BA NaMNAT recently released from the PDB along with two ligated forms. Comparison of these and other less related bacterial NaMNAT structures support the presence of considerable conformational heterogeneity and flexibility in three loops surrounding the substrate-binding area.
doi:10.1107/S1744309108029102
PMCID: PMC2564882  PMID: 18931430
nicotinic acid mononucleotide adenylyltransferase; Bacillus anthracis; self-interaction chromatography; second virial coefficient
7.  Synthesis of Biotin Tagged Chemical Cross-linkers and Their Applications for Mass Spectrometry 
Chemical cross-linking combined with mass spectrometry has been used to elucidate protein structures and protein-protein interactions. However, heterogeneity of the samples and the relatively low abundance of cross-linked peptides make this approach challenging. As an effort to overcome this hurdle, we have synthesized lysine reactive homobifunctional cross-linkers with the biotin in the middle of the linker and used them for enriching cross-linked peptides. The reaction of biotin tagged cross-linkers with purified HIV-1 CA resulted in the formation of hanging and intra-molecular cross-links. The peptides modified with biotinylated cross-linkers were effectively enriched and recovered using a streptavidin coated plate and mass spectrometry friendly buffers. The enrichment of modified peptides and removal of the dominantly unmodified peptides simplify mass spectra and their analyses. The combination of high mass accuracy of FT-ICR MS and MS/MS capability of linear ion trap allows us to unambiguously identify the cross-linking sites and additional modification, such as oxidation.
doi:10.1002/rcm.4066
PMCID: PMC2748246  PMID: 19412923
8.  The Novel Retinoid, 9cUAB30, Inhibits Telomerase and Induces Apoptosis in HL60 Cells1 
Translational Oncology  2008;1(3):148-152.
Telomerase, a ribonucleoprotein important to neoplastic immortality, is up-regulated in approximately 85% of cancers, including leukemias. In this study, 9cUAB30, a novel retinoic acid, resulted in differentiation of HL60 leukemia cells as indicated by morphologic changes characteristic of granulocytes. It also caused a down-regulation of hTERT gene expression and a decrease in telomerase activity. Telomerase inhibition was followed by loss of proliferative capacity, induction of apoptosis, and partial differentiation. These findings demonstrate the effectiveness of 9cUAB30 at inhibiting telomerase activity by down-regulating hTERT gene expression in human leukemic cells.
PMCID: PMC2533143  PMID: 18795149

Results 1-8 (8)