Synthesis of Phenanthro[9,10-c]thiophenes by 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)-Promoted Cyclo-Oxidation.

A new method to prepare phenanthro[9,10-c]thiophenes has been developed. In the presence of triflic acid, 3,4-diaryl thiophenes undergo 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)-promoted cyclo-oxidation. NMR and computational studies indicate that protonation of the thiophene plays a key role in this reaction. The reaction can be used to prepare phenanthro[9,10-c]thiophene, as well as derivatives with alkyl, bromo, and methoxy substituents. However, the yields and selectivity of the reaction depend on both the nature and location of the substituents. Bis(3-methoxyphenyl)thiophene reacts under these conditions to give the desired product in 57 % yield, while bis(4-methoxyphenyl)thiophene gives no product. Bis(3-bromophenyl)thiophene did not react, but cyclo-oxidation of bis(4-bromophenyl)thiophene provides the desired product in 34 % yield.

Progression from latent infection to active disease in dynamic tuberculosis transmission models: a systematic review of the validity of modelling assumptions.

Mathematical modelling is commonly used to evaluate infectious disease control policy and is influential in shaping policy and budgets. Mathematical models necessarily make assumptions about disease natural history and, if these assumptions are not valid, the results of these studies can be biased. We did a systematic review of published tuberculosis transmission models to assess the validity of assumptions about progression to active disease after initial infection (PROSPERO ID CRD42016030009). We searched PubMed, Web of Science, Embase, Biosis, and Cochrane Library, and included studies from the earliest available date (Jan 1, 1962) to Aug 31, 2017. We identified 312 studies that met inclusion criteria. Predicted tuberculosis incidence varied widely across studies for each risk factor investigated. For population groups with no individual risk factors, annual incidence varied by several orders of magnitude, and 20-year cumulative incidence ranged from close to 0% to 100%. A substantial proportion of modelled results were inconsistent with empirical evidence: for 10-year cumulative incidence, 40% of modelled results were more than double or less than half the empirical estimates. These results demonstrate substantial disagreement between modelling studies on a central feature of tuberculosis natural history. Greater attention to reproducing known features of epidemiology would strengthen future tuberculosis modelling studies, and readers of modelling studies are recommended to assess how well those studies demonstrate their validity.

Role of Molecular Recognition in l-Cystine Crystal Growth Inhibition.

l-Cystine kidney stones-aggregates of single crystals of the hexagonal form of l-cystine-afflict more than 20 000 individuals in the United States alone. Current therapies are often ineffective and produce adverse side effects. Recognizing that the growth of l-cystine crystals is a critical step in stone pathogenesis, real-time in situ atomic force microscopy of growth on the (0001) face of l-cystine crystals and measurements of crystal growth anisotropy were performed in the presence of prospective inhibitors drawn from a 31-member library. The most effective molecular imposters for crystal growth inhibition were l-cystine mimics (aka molecular imposters), particularly l-cystine diesters and diamides, for which a kinetic analysis revealed a common inhibition mechanism consistent with Cabrera-Vermilyea step pinning. The amount of inhibitor incorporated by l-cystine crystals, estimated from kinetic data, suggests that imposter binding to the {0001} face is less probable than binding of l-cystine solute molecules, whereas imposter binding to {101̅0} faces is comparable to that of l-cystine molecules. These estimates were corroborated by computational binding energies. Collectively, these findings identify the key structural factors responsible for molecular recognition between molecular imposters and l-cystine crystal kink sites, and the inhibition of crystal growth. The observations are consistent with the reduction of l-cystine stone burden in mouse models by the more effective inhibitors, thereby articulating a strategy for stone prevention based on molecular design.

Regioselective Synthesis of Substituted Cyclopenta[l]phenanthrenes.

A simple and efficient synthesis of cyclopenta[l]phenanthrenes from substituted acetophenones provides access to polycyclic aromatics with a variety of substitution patterns. The synthesis requires only three steps from a silyl enol ether: a Mukaiyama aldol reaction followed by McMurry coupling and then Mallory photocyclooxidation to give the target phenanthrenes. Photocyclization conditions have been found that give regioselective formation of 2,7-phenanthrenes from bis(meta-substituted) stilbenes.

Inhibition of envelope-mediated CD4+-T-cell depletion by human immunodeficiency virus attachment inhibitors.

Human immunodeficiency virus type 1 (HIV-1) envelope (Env) binding induces proapoptotic signals in CD4(+) T cells without a requirement of infection. Defective virus particles, which represent the majority of HIV-1, usually contain a functional Env and therefore represent a potentially significant cause of such CD4(+)-T-cell loss. We reasoned that an HIV-1 inhibitor that prohibits Env-host cell interactions could block the destructive effects of defective particles. HIV-1 attachment inhibitors (AIs), which potently inhibit Env-CD4 binding and subsequent downstream effects of Env, display low-nanomolar antiapoptotic potency and prevent CD4(+)-T-cell depletion from mixed lymphocyte cultures, also with low-nanomolar potency. Specific Env amino acid changes that confer resistance to AI antientry activity eliminate AI antiapoptotic effects. We observed that CD4(+)-T-cell destruction is specific for CXCR4-utilizing HIV-1 strains and that the fusion blocker enfuvirtide inhibits Env-mediated CD4(+)-T-cell killing but is substantially less potent than AIs. These observations, in conjunction with observed antiapoptotic activities of soluble CD4 and the CXCR4 blocker AMD3100, suggest that this AI activity functions through a mechanism common to AI antientry activity, e.g., prevention of Env conformation changes necessary for specific interactions with cellular factors that facilitate viral entry. Our study suggests that AIs, in addition to having potent antientry activity, could contribute to immune system homeostasis in individuals infected with HIV-1 that can engage CXCR4, thereby mitigating the increased risk of adverse clinical events observed in such individuals on current antiretroviral regimens.

Multiplexed assay panel of cytotoxicity in HK-2 cells for detection of renal proximal tubule injury potential of compounds.

Proximal tubules of the kidneys are one of the most common targets of nephrotoxic drugs and chemicals. Screens to predict nephrotoxic potential of compounds with insights to mechanisms of toxicity facilitate lead optimization, guide structure-activity relationships, minimize risks of clinical nephrotoxicity and therefore are valuable in the process of drug discovery. We developed and characterized an in vitro assay multiplexed to measure several endpoints of cytotoxicity using HK-2 cells. Assays for lactate dehydrogenase, cellular caspase 3/7 activation, resazurin dye reduction and Hoechst 33342 DNA staining were multiplexed to maximize the ability to detect cell injury. Assays were performed after 5- or 24-h incubations to further enhance the sensitivity of detection of toxicity. Individual assays were optimized for cell density, assay linearity and assay performance under multiplexed conditions. Inducers of apoptosis (staurosporine) and necrosis (perhexiline) were used to validate the mechanistic aspects of cell death. Nephrotoxic compounds (5-fluorouracil, gentamicin, cisplatin, acetaminophen, para-aminophenol, potassium dichromate, ibuprofen, doxorubicin, cyclosporine, citrinin, puromycin) were used to determine the potential of this method to detect proximal tubule toxicity of compounds. Overall, this cost-effective multiplexed platform is more sensitive than a single endpoint assay, provides mechanistic cues of toxicity and is amenable for higher throughput screening.

Multiplexing a high-throughput liability assay to leverage efficiencies.

In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

Use of cryopreserved transiently transfected cells in high-throughput pregnane X receptor transactivation assay.

Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r(2) > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.