Identification of novel small molecule inhibitors of adenovirus gene transfer using a high throughput screening approach

Due to many favourable attributes adenoviruses (Ads) are the most extensively used vectors for clinical gene therapy applications. However, following intravascular administration, the safety and efficacy of Ad vectors are hampered by the strong hepatic tropism and induction of a potent immune response. Such effects are determined by a range of complex interactions including those with neutralising antibodies, blood cells and factors, as well as binding to native cellular receptors (coxsackie adenovirus receptor (CAR), integrins). Once in the bloodstream, coagulation factor X (FX) has a pivotal role in determining Ad liver transduction and viral immune recognition. Due to difficulties in generating a vector devoid of multiple receptor binding motifs, we hypothesised that a small molecule inhibitor would be of value. Here, a pharmacological approach was implemented to block adenovirus transduction pathways. We developed a high throughput screening (HTS) platform to identify the small molecule inhibitors of FX-mediated Ad5 gene transfer. Using an in vitro fluorescence and cell-based HTS, we evaluated 10,240 small molecules. Following sequential rounds of screening, three compounds, T5424837, T5550585 and T5660138 were identified that ablated FX-mediated Ad5 transduction with low micromolar potency. The candidate molecules possessed common structural features and formed part of the one pharmacophore model. Focused, mini-libraries were generated with structurally related molecules and in vitro screening revealed novel hits with similar or improved efficacy. The compounds did not interfere with Ad5:FX engagement but acted at a subsequent step by blocking efficient intracellular transport of the virus. In vivo, T5660138 and its closely related analogue T5660136 significantly reduced Ad5 liver transgene expression at 48 h post-intravenous administration of a high viral dose (1 × 10<sup>11</sup> vp/mouse). Therefore, this study identifies novel and potent small molecule inhibitors of the Ad5 transduction which may have applications in the Ad gene therapy setting

Regioselective reactions of 3,4-pyridynes enabled by the aryne distortion model.

The pyridine heterocycle continues to play a vital role in the development of human medicines. More than 100 currently marketed drugs contain this privileged unit, which remains highly sought after synthetically. We report an efficient means to access di- and trisubstituted pyridines in an efficient and highly controlled manner using transient 3,4-pyridyne intermediates. Previous efforts to employ 3,4-pyridynes for the construction of substituted pyridines were hampered by a lack of regiocontrol or the inability to later manipulate an adjacent directing group. The strategy relies on the use of proximal halide or sulfamate substituents to perturb pyridyne distortion, which in turn governs regioselectivities in nucleophilic addition and cycloaddition reactions. After trapping of the pyridynes generated in situ, the neighbouring directing groups may be removed or exploited using versatile metal-catalysed cross-coupling reactions. This methodology now renders 3,4-pyridynes as useful synthetic building blocks for the creation of highly decorated derivatives of the medicinally privileged pyridine heterocycle

Discovering Novel Gas-phase Nitrogen-heterocycle Formation Pathways With An Ab Initio Nanoreactor

Nitrogen-containing heterocycles pose an intriguing astrochemical mystery. 45 different varieties have been detected on meteorites with non-terrestrial isotopic abundances, but none have been detected in space, despite numerous search attempts. It is unclear if these species are more likely to form through low-temperature gas-phase chemistry, photoprocessing of icy grains, aqueous chemistry in a meteorite parent body, or a combination of processes. Further exploration of this question requires a broader catalog of potential N-heterocycle precursors, particularly for low temperature gas-phase chemistry. Here, results of \textit{ab initio} molecular dynamics simulations of gas-phase N-heterocycle forming reactions will be discussed. These simulations were performed with an \textit{ab initio} nanoreactor, a computational tool developed for chemical reaction discovery. Multiple novel gas-phase N-heterocycle formation pathways have been revealed, and many reactants are similar to known interstellar molecules. These simulations also reinforce previous experimental and theoretical studies which demonstrated that smaller N-heterocycles are potential precursors to larger ones. While these simulations cannot provide direct insight into interstellar chemistry, they have revealed nonintuitive N-heterocycle precursors that warrant further experimental and astronomical study

Multi-Omic Profiling of Melophlus Sponges Reveals Diverse Metabolomic and Microbiome Architectures that Are Non-overlapping with Ecological Neighbors.

Marine sponge holobionts, defined as filter-feeding sponge hosts together with their associated microbiomes, are prolific sources of natural products. The inventory of natural products that have been isolated from marine sponges is extensive. Here, using untargeted mass spectrometry, we demonstrate that sponges harbor a far greater diversity of low-abundance natural products that have evaded discovery. While these low-abundance natural products may not be feasible to isolate, insights into their chemical structures can be gleaned by careful curation of mass fragmentation spectra. Sponges are also some of the most complex, multi-organismal holobiont communities in the oceans. We overlay sponge metabolomes with their microbiome structures and detailed metagenomic characterization to discover candidate gene clusters that encode production of sponge-derived natural products. The multi-omic profiling strategy for sponges that we describe here enables quantitative comparison of sponge metabolomes and microbiomes to address, among other questions, the ecological relevance of sponge natural products and for the phylochemical assignment of previously undescribed sponge identities

A Second-Generation Janus Scorpionate Ligand: Controlling Coordination Modes in Iron(II) Complexes by Steric Modulation

The second-generation Janus scorpionate ligand [HB(mtdaMe)3−] containing methyl-mercaptothiadiazolyl (mtdaMe) heterocyclic rings and (N,N,N-) and (S,S,S-) binding pockets has been prepared. The effect of methyl substitution versus the unsubstituted first-generation Janus scorpionate [HB(mtda)3]− on the coordination chemistry with alkali metals and on the binding preferences and on the ground spin state of iron(II) complexes has been studied structurally and by 57Fe Mossbauer Spectroscopy

Organic indicators of alteration in the CR chondrites

A study of the organic components in the CR chondrite macromolecule in order to assess the role of pre-terrestrial alteration on the organic inventory

Radiolysis of Solid-State Nitrogen Heterocycles Provides Clues to Their Abundance in the Early Solar System

We studied the radiolysis of a wide variety of N-heterocycles, including many of biological importance, and find that the majority are remarkably stable in the solid-state when subjected to large doses of ionizing gamma radiation from a 60Co source. Degradation of N-heterocycles as a function of dose rate and total dose was measured using high performance liquid chromatography with UV detection. Many N-heterocycles show little degradation when γ-irradiated up to a total dose of ~1 MGy, which approximates hundreds of millions of years’ worth of radiation emitted in meteorite parent bodies due to slow radionuclide decay. Extrapolation of these results suggests that these N-heterocyclic compounds would be stable in dry parent bodies over solar system time-scales. We suggest that the abundance of these N-heterocycles as measured presently in carbonaceous meteorites is largely reflective of their abundance at the time aqueous alteration stopped in their parent bodies, and the absence of certain compounds in present-day samples is either due to the formation mechanisms or degradation which occurred during periods of aqueous alteration or thermal metamorphism

Investigating Hydrothermal and Radiation Effects on Nitrogen Heterocycles Relevant to Meteorite Parent Bodies

Organic compounds in meteorites were likely transformed by a variety of processes on the asteroid parent body including aqueous, thermal, and radiolytic alteration. Previous studies have identified a suite of purine nitrogen heterocycles in carbonaceous chondrites (a class of meteorites) and determined that their likely origin was due to cyanide chemistry. The thesis research described here consisted of two parts: investigating thermal effects on aqueous ammonium cyanide reactions and the production/survivability of organics (Chapter 2) and investigating gamma radiation effects on purine nitrogen heterocycles to understand how prolonged radiation exposure influenced the distribution and abundance of nitrogen heterocycles measured in meteorites today (Chapter 3). A temperature study of 1 M ammonium cyanide was conducted from room temperature to 200 °C (temperatures similar to the aqueous alteration of some carbonaceous chondrites) using a high-pressure reaction vessel. The resulting liquid supernatant and water insoluble cyanide (hetero)polymer were isolated and analyzed by attenuated total reflectance Fourier transform infrared spectroscopy and thermochemolysis gas chromatography-mass spectrometry using tetramethylammonium hydroxide or high-performance liquid chromatography with UV detection (HPLC-UV). A strong correlation was observed between thermochemolysis products of cyanide polymers and the initial reaction temperature - production of aromatic compounds increases and nitrogen containing compounds decrease with respect to increasing reaction temperature. The data presented in this thesis suggests that the polymer may be a less efficient source of nitrogen heterocycles when produced at high temperatures. HPLC-UV analysis showed that the supernatant of heated NH4CN reactions is a complex mixture containing many unknown UV-absorbing chromophores. In addition, multiple nitrogen heterocycles were tentatively identified in the supernatant of heated NH4CN reactions including the high temperature 200 °C reactions. Finally, HPLC was used to determine decomposition of seven purine nitrogen heterocycles exposed to gamma radiation from a cobalt-60 source in an effort to extrapolate abundances during the early formation of the Solar System. Generally speaking, purine nitrogen heterocycles in the solid state are very stable to high doses of γ-radiation; however, guanine experienced a 53% decomposition over the course of ~1 MGy of radiation dose. The radiolysis study correlates to similar conditions in asteroids after their initial aqueous alteration period (i.e. dry alteration) and suggests that meteorite abundances for some compounds (such as adenine) may have remained relatively unchanged over time, but other compounds (like guanine) may have had “original” abundances greater than those currently observed in carbonaceous chondrites

Edge modification of PAHs: the effect of embedded heterocycles on the aromaticity pattern

International audienceAromaticity studies carried out on the condensed model system 2 with different heteroatoms showed that the NICS(1) aromaticity in the five-membered ring correlates with that in the parent five-membered ring having the same heteroelement. Although the local aromaticity pattern is clearly determined by the Clar structure, as evidenced by the local aromaticity values in the reference molecules R1-3, the modifying effect of the heteroatom is significant. The correlation between the NICS(1)5 and NICS(1)6 aromaticities of the neighbouring rings is excellent, by modification of the heterocycle, we can engineer the aromaticity of the connecting six-membered ring as well. Geometry based aromaticity indices do not correlate well with NICS values probably due to annellation effects between rings with different aromatic character. Calculations with the different tested basis sets don’t show significant differences, therefore the use of the computationally cheaper methods is envisaged during further investigations of corresponding systems with extended π-framework