Mary J. Tanga

A Systematic Screen of FDA-Approved Drugs for Inhibitors of Biological Threat Agents

Background The rapid development of effective medical countermeasures against potential biological threat agents is vital. Repurposing existing drugs that may have unanticipated activities as potential countermeasures is one way to meet this important goal, since currently approved drugs already have well-established safety and pharmacokinetic profiles in patients, as well as manufacturing and distribution networks. Therefore, approved drugs could rapidly be made available for a new indication in an emergency. Methodology/Principal Findings A large systematic effort to determine whether existing drugs can be used against high containment bacterial and viral pathogens is described. We assembled and screened 1012 FDA-approved drugs for off-label broad-spectrum efficacy against Bacillus anthracis; Francisella tularensis; Coxiella burnetii; and Ebola, Marburg, and Lassa fever viruses using in vitro cell culture assays. We found a variety of hits against two or more of these biological threat pathogens, which were validated in secondary assays. As expected, antibiotic compounds were highly active against bacterial agents, but we did not identify any non-antibiotic compounds with broad-spectrum antibacterial activity. Lomefloxacin and erythromycin were found to be the most potent compounds in vivo protecting mice against Bacillus anthracis challenge. While multiple virus-specific inhibitors were identified, the most noteworthy antiviral compound identified was chloroquine, which disrupted entry and replication of two or more viruses in vitro and protected mice against Ebola virus challenge in vivo. Conclusions/Significance The feasibility of repurposing existing drugs to face novel threats is demonstrated and this represents the first effort to apply this approach to high containment bacteria and viruses.

A parallel chiral-achiral liquid chromatographic method for the determination of the stereoisomers of ketamine and ketamine metabolites in the plasma and urine of patients with complex regional pain syndrome

A parallel chiral/achiral LC-MS/MS assay has been developed and validated to measure the plasma and urine concentrations of the enantiomers of ketamine, (R)- and (S)-Ket, in complex regional pain syndrome (CRPS) patients receiving a 5-day continuous infusion of a sub-anesthetic dose of (R,S)-Ket. The method was also validated for the determination of the enantiomers of the Ket metabolites norketamine, (R)- and (S)-norKet and dehydronorketamine, (R)- and (S)-DHNK, as well as the diastereomeric metabolites hydroxynorketamine, (2S,6S)-/(2R,6R)-HNK and two hydroxyketamines, (2S,6S)-HKet and (2S,6R)-Hket. In this method, (R,S)-Ket, (R,S)-norKet and (R,S)-DHNK and the diastereomeric hydroxyl-metabolites were separated and quantified using a C(18) stationary phase and the relative enantiomeric concentrations of (R,S)-Ket, (R,S)-norKet and (R,S)-DHNK were determined using an AGP-CSP. The analysis of the results of microsomal incubations of (R)- and (S)-Ket and a plasma and urine sample from a CRPS patient indicated the presence of 10 additional compounds and glucuronides. The data from the analysis of the patient sample also demonstrated that a series of HNK metabolites were the primary metabolites in plasma and (R)- and (S)-DHNK were the major metabolites found in urine. The results suggest that norKet is the initial, but not the primary metabolite and that downstream norKet metabolites play a role in (R,S)-Ket-related pain relief in CRPS patients.

Comparative Molecular Field Analysis of Fenoterol Derivatives: A Platform Towards Highly Selective and Effective β2 Adrenergic Receptor Agonists

PURPOSE To use a previously developed CoMFA model to design a series of new structures of high selectivity and efficacy towards the beta(2)-adrenergic receptor. RESULTS Out of 21 computationally designed structures 6 compounds were synthesized and characterized for beta(2)-AR binding affinities, subtype selectivities and functional activities. CONCLUSION the best compound is (R,R)-4-methoxy-1-naphthylfelnoterol with K(i)beta(2)-AR=0.28microm, K(i)beta(1)-AR/K(i)beta(2)-AR=573, EC(50cAMP)=3.9nm, EC(50cardio)=16nm. The CoMFA model appears to be an effective predictor of the cardiomocyte contractility of the studied compounds which are targeted for use in congestive heart failure.

Repurposing FDA-approved drugs to combat drug-resistant Acinetobacter baumannii

OBJECTIVE The rising occurrence of drug-resistant pathogens accentuates the need to identify novel antibiotics. We wanted to identify new scaffolds for drug discovery by repurposing FDA-approved drugs against Acinetobacter baumannii, an emerging Gram-negative nosocomial drug-resistant pathogen. MATERIALS AND METHODS In this study, we screened 1040 FDA-approved drugs against drug-susceptible A. baumannii ATCC 17978 and drug-resistant A. baumannii BAA-1605. RESULTS AND DISCUSSION Twenty compounds exhibited significant antimicrobial activity (MIC ≤8 mg/L) against ATCC 17978 while only five compounds showed such activity against BAA-1605. Among the most notable results, tyrothricin, a bactericidal antibiotic typically active only against Gram-positive bacteria, exhibited equipotent activity against both strains. CONCLUSION The paucity of identified compounds active against drug-resistant A. baumannii exemplifies its ability to resist antimicrobials as well as the resilience of drug-resistant Gram-negative pathogens. Repurposing of approved drugs is a viable alternative to de novo drug discovery and development.

Diamino-1,2,4-triazole derivatives are selective inhibitors of TYK2 and JAK1 over JAK2 and JAK3

Tyrosine kinase 2 (TYK2) is required for signaling of interleukin-23 (IL-23), which plays a key role in rheumatoid arthritis. Presented is the design and synthesis of 1,2,4-triazoles, and the evaluation of their inhibitory activity against the Janus associated kinases TYK2 and JAKs 1-3.

Tyrosine 308 Is Necessary for Ligand-directed Gs Protein-biased Signaling of β2-Adrenoceptor

Background: Ligand-specific receptor signaling is often referred to as functional selectivity or biased agonism. Results: Single amino acid substitution on β2-adrenoreceptor (Y308F) converts a ligand-specific signaling from Gs-biased to promiscuous Gs and Gi dual signaling. Conclusion: Specific ligand-receptor interaction results in receptor conformation(s) sufficient to convey biased signaling. Significance: Our work reveals a molecular mechanism for biased agonism. Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, β2-adrenoceptor (β2-AR) couples dually to Gs and Gi proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of β2-AR causes a switch in receptor coupling from Gs to Gi. More recent studies have demonstrated that phosphorylation of β2-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the Gi coupling. We have previously shown that although most β2-AR agonists cause both Gs and Gi activation, (R,R′)-fenoterol preferentially activates β2-AR-Gs signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on β2-AR essential for Gs-biased signaling. Following stimulation with a β2-AR-Gs-biased agonist (R,R′)-4′-aminofenoterol, the Gi disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type β2-AR. Interestingly, Y308F substitution on β2-AR enabled (R,R′)-4′-aminofenoterol to activate Gi and to produce these responses in a pertussis toxin-sensitive manner without altering β2-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of β2-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of β2-AR stabilize receptor conformations favoring the receptor-Gs protein coupling and subsequently result in Gs-biased agonism.

Polycyclic Aromatic Nitrogen Heterocycles. Part II: Effect of Solvent Polarity on the Fluorescence Emission Fine Structure of Three Azapyrene Compounds

Fluorescence emission spectra are reported for 1-azapyrene, 2-azapyrene, and 4-azapyrene dissolved in several nonelectrolyte solvents of varying polarity. Results of these measurements show that all three solutes are unsuitable for solvent polarity probes, as the calculated emission intensity ratios remain nearly constant, irrespective of solvent polarity. Abnormal fluorescence behavior was also noted for 1-azapyrene and 2-azapyrene dissolved in several chlorinated solvents. Time-dependent fluorescence spectra for 1-azapyrene in dichloromethane suggest a possible photochemical reaction between the PANH solute and chlorinated solvent molecule.

Polycyclic Aromatic Nitrogen Heterocycles. Part III: Effect of Solvent Polarity and Solvent Acidity on the Fluorescence Emission Behavior of Select Azapyrenes and Phenanthroisoquinolines

Fluorescence emission spectra are reported for 1-azapyrene, 2-azapyrene, 4-azapyrene, 12-azabenzo[a|pyrene, phenanthro[9,10g]isoquinoline, phenanthro[2,3h|isoquinoline, and phenanthro[3,2h]isoquinoline dissolved in organic nonelectrolyte solvents of varying polarity and acidity. Results of these measurements indicate that 12-azabenzo[a]pyrene, phenanthro[2,3h]isoquinoline, and phenanthro[3,2h]isoquinoline exhibit modest probe character as evidenced by decreased I/II or I/III band emission intensity ratios with increasing solvent polarity. Alcoholic solvents such as trifluoroethanol protonate the nitrogen hetero-atom, resulting in loss of emission fine structure accompanied by a sizeable red-shift in emission wavelength(s). For 1-azapyrene and 2-azapyrene the observed fluorescence emission spectra showed both neutral and protonated forms of the solute in trifluoroethanol.

Effect of Solvent Polarity and Acidity on Fluorescence Emission Fine Structures of Select Aza-Polynuclear Aromatics and Dibenzo[b,n]Perylene Hetero-Atom Derivatives

Fluorescence emission spectra are reported for 1-azabenz[a]anthracene, 2-azabenz[a]anthracene, 9-azabenz[a]anthracene, 4-azachrysene, 1,2,7,8-tetraazacoronene, dibenzo[2,3:10,11]perylo[1,12bcd]furan, dibenzo[2,3:10,11]perylo[1,12bcd]thiophene, and dibenzo[2,3:10,11]perylo[1,12bcd] selenophene dissolved in various organic solvents. Results of these measurements are used to classify the solutes into probe or nonprobe molecules depending upon whether emission intensity ratios vary systematically with solvent polarity. Of the eight aromatic solutes examined, only the three azabenzanthracenes showed slight probe character.

Polycyclic Aromatic Nitrogen Heterocycles. Part IV: Effect of Solvent Polarity, Solvent Acidity, Nitromethane and 1,2,4-Trimethoxybenzene on the Fluorescence Emission Behavior of Select Monoaza- and Diazaarenes

Fluorescence emission spectra are reported for naphth[2′l′8′7′: 4,10,5]anthra[l,9,8cdef]cinnoline, benzo[lmn][3,8]phenanthroline (also called 2,7-diazapyrene), benz[4,10]anthra[l,9,8cdef]cinnoline, naphtho[8,1,2hij]pyreno[9,10,ldef]phthalazine, acenaphtho[l,2b]pyridine, benzo[a]phenazine, indeno[l,2,3ij][2,7]naphthyridine, and indeno-[l,2,3ij]isoquinoline dissolved in organic nonelectrolyte solvents of varying polarity and acidity. Results of these measurements indicate that naphth[2′,1′,8′,7′:4,10,5]anthra[l,9,8cdef]cinnoline exhibits some signs of probe character as evidenced by changing emission intensity ratios; however, numerical values did not vary systematically with solvent polarity. The effect of nitromethane and 1,2,4-trimethoxybenzene as selective quenching agents on both the unprotonated and protonated PANHs was also examined. Nitromethane was found to quench fluorescence emission of roughly two-thirds of the alternant unprotonated PANHs studied to date. Emission intensities of the protonated PANHs remained essentially constant and were not affected by nitromethane. 1,2,4-Trimethoxybenzene, on the other hand, quenched the fluorescence emission of several unprotonated and all protonated PANHs examined.