Unconventional surface plasmon resonance signals reveal quantitative inhibition of transcriptional repressor EthR by synthetic ligands

International audienceEthR is a mycobacterial repressor that limits the bioactivation of ethionamide, a commonly used anti-tuberculosis second-line drug. Several efforts have been deployed to identify EthR inhibitors abolishing the DNA-binding activity of the repressor. This led to the demonstration that stimulating the bioactivation of ETH through EthR inhibition could be an alternative way to fight Mycobacterium tuberculosis. We propose a new SPR methodology to study the affinity between inhibitors and EthR. Interestingly, the binding between inhibitors and immobilized EthR produced a dose dependent negative SPR signal. We demonstrated that this signal reveals the affinity of the small molecules for the repressor. The affinity constants (KD) correlated with their capacity to inhibit the binding of EthR to DNA. We hypothesize that conformational changes of EthR during ligand interaction could be responsible for this SPR signal. Practically, this unconventional result open perspectives to the development of SPR assay that would at the same time tough on the structural changes of the target upon binding with an inhibitor and on the binding constant of this interaction

Evaluation of critical parameters in the hollow-fibre system for tuberculosis: A case study of moxifloxacin

AimsThe hollow‐fibre system for tuberculosis (HFS‐TB) is a preclinical model qualified by the European Medicines Agency to underpin the anti‐TB drug development process. It can mimic in vivo pharmacokinetic (PK)–pharmacodynamic (PD) attributes of selected antimicrobials, which could feed into in silico models to inform the design of clinical trials. However, historical data and published protocols are insufficient and omit key information to allow experiments to be reproducible. Therefore, in this work, we aim to optimize and standardize various HFS‐TB operational procedures.MethodsFirst, we characterized bacterial growth dynamics with different types of hollow‐fibre cartridges, Mycobacterium tuberculosis strains and media. Second, we mimicked a moxifloxacin PK profile within hollow‐fibre cartridges, in order to check drug–fibres compatibility. Lastly, we mimicked the moxifloxacin total plasma PK profile in human after once daily oral dose of 400 mg to assess PK–PD after different sampling methods, strains, cartridge size and bacterial adaptation periods before drug infusion into the system.ResultsWe found that final bacterial load inside the HFS‐TB was contingent on the studied variables. Besides, we demonstrated that drug–fibres compatibility tests are critical preliminary HFS‐TB assays, which need to be properly reported. Lastly, we uncovered that the sampling method and bacterial adaptation period before drug infusion significantly impact actual experimental conclusions.ConclusionOur data contribute to the necessary standardization of HFS‐TB experiments, draw attention to multiple aspects of this preclinical model that should be considered when reporting novel results and warn about critical parameters in the HFS‐TB currently overlooked

Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands

Ethionamide is an antituberculous drug for the treatment of multidrug-resistant Mycobacterium tuberculosis. This antibiotic requires activation by the monooxygenase EthA to exert its activity. Production of EthA is controlled by the transcriptional repressor EthR, a member of the TetR family. The sensitivity of M. tuberculosis to ethionamide can be artificially enhanced using synthetic ligands of EthR that allosterically inactivate its DNA-binding activity. Comparison of several structures of EthR co-crystallized with various ligands suggested that the structural reorganization of EthR resulting in its inactivation is controlled by a limited portion of the ligand-binding-pocket. In silico simulation predicted that mutation G106W may mimic ligands. X-ray crystallography of variant G106W indeed revealed a protein structurally similar to ligand-bound EthR. Surface plasmon resonance experiments established that this variant is unable to bind DNA, while thermal shift studies demonstrated that mutation G106W stabilizes EthR as strongly as ligands. Proton NMR of the methyl regions showed a lesser contribution of exchange broadening upon ligand binding, and the same quenched dynamics was observed in apo-variant G106W. Altogether, we here show that the area surrounding Gly106 constitutes the molecular switch involved in the conformational reorganization of EthR. These results also shed light on the mechanistic of ligand-induced allosterism controlling the DNA binding properties of TetR family repressors

Intrinsic antibacterial activity of nanoparticles made of β-cyclodextrins potentiates their effect as drug nanocarriers against tuberculosis

Multi-drug-resistant tuberculosis (TB) is a major public health problem, concerning about half a million cases each year. Patients hardly adhere to the current strict treatment consisting of more than 10 000 tablets over a 2-year period. There is a clear need for efficient and better formulated medications. We have previously shown that nanoparticles made of cross-linked poly-β-cyclodextrins (pβCD) are efficient vehicles for pulmonary delivery of powerful combinations of anti-TB drugs. Here, we report that in addition to being efficient drug carriers, pβCD nanoparticles are endowed with intrinsic antibacterial properties. Empty pβCD nanoparticles are able to impair Mycobacterium tuberculosis (Mtb) establishment after pulmonary administration in mice. pβCD hamper colonization of macrophages by Mtb by interfering with lipid rafts, without inducing toxicity. Moreover, pβCD provoke macrophage apoptosis, leading to depletion of infected cells, thus creating a lung microenvironment detrimental to Mtb persistence. Taken together, our results suggest that pβCD nanoparticles loaded or not with antibiotics have an antibacterial action on their own and could be used as a carrier in drug regimen formulations effective against TB.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Efficient, protection-free Suzuki-Miyaura synthesis of ortho-biphenyltetrazoles

We describe an efficient protocol for the Suzuki-Miyaura synthesis of ortho-biphenyltetrazoles from non-protected 2-bromophenyltetrazole and arylboronic acids. The optimised conditions were achieved using [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) as catalyst and Na2CO3 as base. A panel of structurally diverse arylboronic acids was used to demonstrate the scope of the coupling procedure. Keywords: ortho-Biphenyltetrazole; Suzuki-Miyaura cross-coupling; Microwave synthesi

UFU ('Ullmann-Finkelstein-Ullmann'): a new multicomponent reaction

We developed conditions to carry out the first 'one-pot' Ullmann-Finkelstein-Ullmann multicomponent reaction reported. This reaction allows the one-pot synthesis of dissymmetrical para-disubstituted benzene scaffold from 1-bromo-4-iodobenzene and two N-nucleophiles. CuI/N,N'-dimethyl-cyclohexane-1,2-diamine was used as a catalyst/ligand couple, K3PO4 as a base and the reaction was performed in dioxane

Carbohydrate-protein interactions at interfaces: synthesis of thiolactosyl glycolipids and design of a working model for surface plasmon resonance.

Thiolactosyl lipids designed for carbohydrate-protein binding studies have been synthesised. One representative was selected for binding studies with a plant lectin RCA120, the agglutinin from Ricinus communis. The interactions were measured quantitatively in real time using a BIAcore surface plasmon resonance instrument. Removal of much of the galactose from the thiolactosyl lipid in situ with beta-galactosidase showed that the lectin binding was highly specific. A dissociation constant KD = 8.77 x 10(-8) M was measured for 1-[2-[2-(2-[beta-D-galactopyranosyl-(1-->4)-1-thio-beta-D -glucopyranosyl]ethoxy)ethoxy]ethoxy]octadecane 30 which is four orders of magnitude greater than that determined for binding to lactose in solution. A concentration of lactose of > 80 mM was required to block the lectin binding to thiolactosyl lipid in a neomembrane

Synthesis of N-(iodophenyl)-amides via an unprecedented Ullmann-Finkelstein tandem reaction

Using a new Ullmann-Finkelstein tandem reaction, N-(iodophenyl)-amides were synthesized from the corresponding amides and iodo-bromobenzenes. The catalyst/ligand couple CuI/N,N'-dimethyl-cyclohexane-1,2-diamine was used for this reaction in dioxane with K3PO4 as base

Microwave-assisted synthesis of functionalized spirohydantoins as 3-D privileged fragments for scouting the chemical space

International audienc

Synthesis and structural studies of a novel scaffold for drug discovery: a 4,5-dihydro-3H-spiro[1,5-benzoxazepine-2,4'-piperidine]

We describe the three-step synthesis of a novel 4,5-dihydro-3H-spiro[1,5-benzoxazepine-2,4'-piperidine] scaffold from ortho-hydroxyacetophenone and N-benzylpiperidone. The structure of one disubstituted derivative, studied by NOESY NMR in an aqueous medium and X-ray diffraction, demonstrates that this scaffold presents side chains in a well-defined orientation. The Boc protected derivative represents a key intermediate for the combinatorial synthesis of drug-like molecules