Available chemotherapeutic options are very limited against Mycobacterium abscessus, which imparts a particular challenge in the treatment of cystic fibrosis (CF) patients infected with this rapidly growing mycobacterium. New drugs are urgently needed against this emerging pathogen, but the discovery of active chemotypes has not been performed intensively. Interestingly, however, the repurposing of thiacetazone (TAC), a drug once used to treat tuberculosis, has increased following the deciphering of its mechanism of action and the detection of significantly more potent analogues. We therefore report studies performed on a library of 38 TAC-related derivatives previously evaluated for their antitubercular activity. Several compounds, including D6, D15, and D17, were found to exhibit potent activity in vitro against M. abscessus, Mycobacterium massiliense, and Mycobacterium bolletii clinical isolates from CF and non-CF patients. Similar to TAC in Mycobacterium tuberculosis, the three analogues act as prodrugs in M. abscessus, requiring bioactivation by the EthA enzyme, MAB_0985. Importantly, mutations in the transcriptional TetR repressor MAB_4384, with concomitant upregulation of the divergently oriented adjacent genes encoding an MmpS5/MmpL5 efflux pump system, accounted for high cross-resistance levels among all three compounds. Overall, this study uncovered a new mechanism of drug resistance in M. abscessus and demonstrated that simple structural optimization of the TAC scaffold can lead to the development of new drug candidates against M. abscessus infections.

Defining the pharmacological target(s) of currently used drugs and developing new analogues with greater potency are both important aspects of the search for agents that are effective against drug-sensitive and drug-resistant Mycobacterium tuberculosis. Thiacetazone (TAC) is an anti-tubercular drug that was formerly used in conjunction with isoniazid, but removed from the antitubercular chemotherapeutic arsenal due to toxic side effects. However, several recent studies have linked the mechanisms of action of TAC to mycolic acid metabolism and TAC-derived analogues have shown increased potency against M. tuberculosis. To obtain new insights into the molecular mechanisms of TAC resistance, we isolated and analyzed 10 mutants of M. tuberculosis that were highly resistant to TAC. One strain was found to be mutated in the methyltransferase MmaA4 at Gly101, consistent with its lack of oxygenated mycolic acids. All remaining strains harbored missense mutations in either HadA (at Cys61) or HadC (at Val85, Lys157 or Thr123), which are components of the β-hydroxyacyl-ACP dehydratase complex that participates in the mycolic acid elongation step. Separately, a library of 31 new TAC analogues was synthesized and evaluated against M. tuberculosis. Two of these compounds, 15 and 16, exhibited minimal inhibitory concentrations 10-fold lower than the parental molecule, and inhibited mycolic acid biosynthesis in a dose-dependent manner. Moreover, overexpression of HadAB HadBC or HadABC in M. tuberculosis led to high level resistance to these compounds, demonstrating that their mode of action is similar to that of TAC. In summary, this study uncovered new mutations associated with TAC resistance and also demonstrated that simple structural optimization of the TAC scaffold was possible and may lead to a new generation of TAC-derived drug candidates for the potential treatment of tuberculosis as mycolic acid inhibitors.

We synthesise, optically trap and rotate individual nanovaterite crystals with a mean particle radius of 423 nm. Rotation rates of up to 4.9 kHz in heavy water are recorded. Laser-induced heating due to residual absorption of the nanovaterite particle results in the superlinear behaviour of the rotation rate as a function of trap power. A finite element method based on the Navier-Stokes model for the system allows us to determine the residual optical absorption coefficient for a trapped nanovaterite particle. This is further confirmed by the theoretical model. Our data show that the translational Stokes drag force and rotational Stokes drag torque needs to be modified with appropriate correction factors to account for the power dissipated by the nanoparticle.

Lectin-functionalized silver nanoparticles have been successfully designed for use as molecular imaging agents to investigate carbohydrate-lectin interactions at the surface of mammalian cells, using surface-enhanced Raman scattering (SERS). Carbohydrate-lectin interactions are key to many cellular processes and are responsible for controlling an array of cellular interactions. In this study, lectin-functionalized silver nanoparticles were used to detect the expression of carbohydrate species at the cellular interface. The carbohydrate-lectin interactions were demonstrated using three different lectin species for three distinct cell types. Due to the known difference between the expressions of glycans in cancerous versus noncancerous cells of the same origin, this approach has been expanded to study both cancerous and noncancerous prostate cells. This has been achieved via confocal SERS mapping of the expression of the key glycan, sialic acid, on the surface of each of these cell types. In achieving such discrimination, a novel method has been created by which glycan expression can be reproducibly monitored. Comparative studies were performed using both fluorescence and SERS. SERS provided an increased discrimination over fluorescence when analyzing cell subsets to discriminate between cancerous and noncancerous cells. The success of this method means that it could be used to complement the current gold standard histopathological techniques.

An on/off SERS aggregation system has been designed to investigate carbohydrate-lectin interactions. Detection of the lectin ConA was achieved at pico molar levels. Discrimination was also demonstrated between two different carbohydrate moieties demonstrating the specific interaction between the carbohydrate and specific protein present.