Manikandan Kadirvel

Mutation rate plasticity in rifampicin resistance depends on Escherichia coli cell–cell interactions

Variation of mutation rate at a particular site in a particular genotype, in other words mutation rate plasticity (MRP), can be caused by stress or ageing. However, mutation rate control by other factors is less well characterized. Here we show that in wild-type Escherichia coli (K-12 and B strains), the mutation rate to rifampicin resistance is plastic and inversely related to population density: lowering density can increase mutation rates at least threefold. This MRP is genetically switchable, dependent on the quorum-sensing gene luxS—specifically its role in the activated methyl cycle—and is socially mediated via cell–cell interactions. Although we identify an inverse association of mutation rate with fitness under some circumstances, we find no functional link with stress-induced mutagenesis. Our experimental manipulation of mutation rates via the social environment raises the possibility that such manipulation occurs in nature and could be exploited medically.

Boron-Based Inhibitors of the NLRP3 Inflammasome

Summary NLRP3 is a receptor important for host responses to infection, yet is also known to contribute to devastating diseases such as Alzheimers disease, diabetes, atherosclerosis, and others, making inhibitors for NLRP3 sought after. One of the inhibitors currently in use is 2-aminoethoxy diphenylborinate (2APB). Unfortunately, in addition to inhibiting NLRP3, 2APB also displays non-selective effects on cellular Ca2+ homeostasis. Here, we use 2APB as a chemical scaffold to build a series of inhibitors, the NBC series, which inhibit the NLRP3 inflammasome in vitro and in vivo without affecting Ca2+ homeostasis. The core chemical insight of this work is that the oxazaborine ring is a critical feature of the NBC series, and the main biological insight the use of NBC inhibitors led to was that NLRP3 inflammasome activation was independent of Ca2+. The NBC compounds represent useful tools to dissect NLRP3 function, and may lead to oxazaborine ring-containing therapeutics.

A caspase-3 ?death-switch? in colorectal cancer cells for induced and synchronous tumor apoptosis in vitro and in vivo facilitates the development of minimally invasive cell death biomarkers

Novel anticancer drugs targeting key apoptosis regulators have been developed and are undergoing clinical trials. Pharmacodynamic biomarkers to define the optimum dose of drug that provokes tumor apoptosis are in demand; acquisition of longitudinal tumor biopsies is a significant challenge and minimally invasive biomarkers are required. Considering this, we have developed and validated a preclinical ‘death-switch’ model for the discovery of secreted biomarkers of tumour apoptosis using in vitro proteomics and in vivo evaluation of the novel imaging probe [18F]ML-10 for non-invasive detection of apoptosis using positron emission tomography (PET). The ‘death-switch’ is a constitutively active mutant caspase-3 that is robustly induced by doxycycline to drive synchronous apoptosis in human colorectal cancer cells in vitro or grown as tumor xenografts. Death-switch induction caused caspase-dependent apoptosis between 3 and 24 hours in vitro and regression of ‘death-switched’ xenografts occurred within 24 h correlating with the percentage of apoptotic cells in tumor and levels of an established cell death biomarker (cleaved cytokeratin-18) in the blood. We sought to define secreted biomarkers of tumor apoptosis from cultured cells using Discovery Isobaric Tag proteomics, which may provide candidates to validate in blood. Early after caspase-3 activation, levels of normally secreted proteins were decreased (e.g. Gelsolin and Midkine) and proteins including CD44 and High Mobility Group protein B1 (HMGB1) that were released into cell culture media in vitro were also identified in the bloodstream of mice bearing death-switched tumors. We also exemplify the utility of the death-switch model for the validation of apoptotic imaging probes using [18F]ML-10, a PET tracer currently in clinical trials. Results showed increased tracer uptake of [18F]ML-10 in tumours undergoing apoptosis, compared with matched tumour controls imaged in the same animal. Overall, the death-switch model represents a robust and versatile tool for the discovery and validation of apoptosis biomarkers.

Exciplex and excimer molecular probes: detection of conformational flip in a myo-inositol chair.

2-O-tert-Butyldimethylsilyl-4,6-bis-O-pyrenoyl-myo-inositol-1,3,5-orthoformate (6) and 2-O-tert-butyldimethylsilyl-4-O-[4-(dimethylamino)benzoyl]-6-O-pyrenoyl-myo-inositol-1,3,5-orthoacetate (10) adopt conformationally restricted unstable chairs with five axial substituents. In the symmetrical diester 6, the two pi-stacked pyrenoyl groups are electron acceptor-donor partners, giving a strong intramolecular excimer emission. In the mixed ester 10, the pyrenoyl group is the electron acceptor and the 4-(dimethylamino)benzoyl ester is the electron donor, giving a strong intramolecular exciplex emission. The conformation of the mixed ester 10 was assessed using 1H NMR spectroscopy (1H-NOESY) and computational studies. which showed the minimum inter-centroid distance between the two aromatic systems to be approximately 3.9 A. Upon addition of acid, the orthoformate/orthoacetate trigger in 6 and 10 was cleaved, which caused a switch of the conformation of the myo-inositol ring to the more stable penta-equatorial chair, leading to separation of the aromatic ester groups and loss of excimer and exciplex fluorescence, respectively. This study provides proof of principle for the development of novel fluorescent molecular probes.

Detection of apoptosis by PET/CT with the diethyl ester of [18F]ML-10 and fluorescence imaging with a dansyl analogue

The diethyl ester of [(18)F]ML-10 is a small molecule apoptotic PET probe for cancer studies. Here we report a novel multi-step synthesis of the diethyl ester of ML-10 in excellent yields via fluorination using Xtal-Fluor-E. In addition, a one-pot radiosynthesis of the diethyl ester of [(18)F]ML-10 from nucleophilic [(18)F]fluoride was completed in 23% radiochemical yield (decay corrected). The radiochemical purity of the product was ≥99%. The diethyl ester of [(18)F]ML-10 was used in vivo to detect apoptosis in the testes of mice. In parallel studies, the dansyl-ML-10 diethyl ester was prepared and used to detect apoptotic cells in an in vitro cell based assay.

Development & Automation of a novel [18F]F prosthetic group, 2-[18F]-fluoro-3-pyridinecarboxaldehyde, and its application to an amino(oxy)-functionalised Aβ peptide

2-[18F]-Fluoro-3-pyridinecarboxaldehyde ([18F]FPCA) is a novel, water-soluble prosthetic group. Its radiochemistry has been developed and fully-automated for application in chemoselective radiolabelling of amino(oxy)-derivatised RI-OR2-TAT peptide, (Aoa-k)-RI-OR2-TAT, using a GE TRACERlab FX-FN. RI-OR2-TAT is a brain-penetrant, retro-inverso peptide that binds to amyloid species associated with Alzheimers Disease. Radiolabelled (Aoa-k)-RI-OR2-TAT was reproducibly synthesised and the product of the reaction with FPCA has been fully characterised. In-vivo biodistribution of [18F]RI-OR2-TAT has been measured in Wistar rats.

Where antibiotic resistance mutations meet quorum-sensing

We do not need to rehearse the grim story of the global rise of antibiotic resistant microbes. But what if it were possible to control the rate with which antibiotic resistance evolves by de novo mutation? It seems that some bacteria may already do exactly that: they modify the rate at which they mutate to antibiotic resistance dependent on their biological environment. In our recent study [Krašovec, et al. Nat. Commun. (2014), 5, 3742] we find that this modification depends on the density of the bacterial population and cell-cell interactions (rather than, for instance, the level of stress). Specifically, the wild-type strains of Escherichia coli we used will, in minimal glucose media, modify their rate of mutation to rifampicin resistance according to the density of wild-type cells. Intriguingly, the higher the density, the lower the mutation rate (Figure 1). Why this novel density-dependent ‘mutation rate plasticity’ (DD-MRP) occurs is a question at several levels. Answers are currently fragmentary, but involve the quorum-sensing gene luxS and its role in the activated methyl cycle.

Radiosynthesis and reactivity of N-[11C]methyl carbamoylimidazole

N-Methyl carbamoylimidazole is a safe and practical alternative to methyl isocyanate for carbamoylation reactions. We have developed a new chemical route for its synthesis from methyl iodide and applied this to the synthesis of N-[11C]methyl carbamoylimidazole as a new [11C]synthon to radiolabel biomolecules for PET imaging research. N-[11C]methyl carbamoylimidazole was prepared from [11C]methyl iodide in 70–74% radiochemical yield (decay corrected) and can be used in situ for further reaction without purification. The reactivity of N-[11C]methyl carbamoylimidazole was demonstrated in a series of [11C]carbamoylation reactions.

Evaluation of analogues of furan-amidines as inhibitors of NQO2

Inhibitors of the enzyme NQO2 (NRH: quinone oxidoreductase 2) are of potential use in cancer chemotherapy and malaria. We have previously reported that non-symmetrical furan amidines are potent inhibitors of NQO2 and here novel analogues are evaluated. The furan ring has been changed to other heterocycles (imidazole, N-methylimidazole, oxazole, thiophene) and the amidine group has been replaced with imidate, reversed amidine, N-arylamide and amidoxime to probe NQO2 activity, improve solubility and decrease basicity of the lead furan amidine. All compounds were fully characterised spectroscopically and the structure of the unexpected product N-hydroxy-4-(5-methyl-4-phenylfuran-2-yl)benzamidine was established by X-ray crystallography. The analogues were evaluated for inhibition of NQO2, which showed lower activity than the lead furan amidine. The observed structure-activity relationship for the furan-amidine series with NQO2 was rationalized by preliminary molecular docking and binding mode analysis. In addition, the oxazole-amidine analogue inhibited the growth of Plasmodium falciparum with an IC50 value of 0.3 μM.

Radiolabelling of an anti-amyloid peptide with 6-(2-[18F]Fluoroethoxy)-2-cyanobenzothiazole

Background Many nuclear medicine departments have an extensive radiopharmaceutical portfolio. Consequently, these multiple PET radiopharmaceuticals have to be produced with the same synthesis module. An important consideration in GMP-compliant PET production is to avoid potential cross-contamination. Flexible cassette-based synthesis modules with disposable components could provide an attractive solution to overcome this hurdle. Because only disposable materials are used, validation of cleaning procedures is not required and maintenance of the system can be reduced. For this purpose, we aimed to develop cassette-based synthesis methods for a variety of11C and18F labeled PET radiopharmaceuticals. Methods In our study, the production processes for the radiosynthesis of [11C]choline, [11C]methionine, [11C]PK11195, [18F]CP18, [18F]FDHT and [18F]FES were implemented on the cassette-based Eckert & Ziegler PharmTracer synthesis module. In order to achieve this goal, there is a need for each tracer to develop a dedicated design of its corresponding cassette. The radiopharmaceuticals were validated according to GMP guidelines and compliance to the required pharmaceutical criteria was tested including novel UPLC analytical QC methods. Results Radiochemical yields obtained with the cassette based system were comparable to literature; [11C]choline 28-52%; [11C]methionine 24-39%; [11C]PK11195 9-15%, [18F]CP18 7-13%, [18F]FDHT 5-10%, [18F]FES 16-28%. Synthesis time was 25 min for [11C]choline and [11C]methionine, 55 min for [11C]PK11195 and 2 h for the 18Flabeled radiopharmaceuticals. All radiopharmaceuticals meets the requirements for specific activities and were >95% pure, isotonic, and complied to the prospective specification for endotoxins, sterility and organic solvents. Discussion and conclusion Multistep synthesis procedures for PET radiopharmaceuticals, involving11C-methylation,11F-fluorination, click chemistry, distillation and solid phase extraction, are feasible with the used cassette-based synthesis module. In addition, purification with HPLC and formulation of the radiopharmaceuticals were successfully performed. The design of the cassettes and the synthesis programs of individual radiopharmaceuticals can easily be adapted for the production of other radiopharmaceuticals, which makes the system very flexible. All cassettes and other materials are disposable; therefore no cross-contamination is possible between the productions of different radiopharmaceuticals. In conclusion, our results show that a variety of11C and11F-radiopharmaceuticals can be reliably produced under GMP-compliant conditions using a common synthesis module with disposable cassettes.