Rational Design and Synthesis of Thioridazine Analogues as Enhancers of the Antituberculosis Therapy

Tuberculosis, caused by <i>Mycobacterium tuberculosis</i>, is still one of the leading infectious diseases globally. Therefore, novel approaches are needed to face this disease. Efflux pumps are known to contribute to the emergence of <i>M. tuberculosis</i> drug resistance. Thioridazine has shown good anti-TB properties both in vitro and in vivo, likely due to its capacity to inhibit efflux mechanisms. Here we report the design and synthesis of a number of putative efflux inhibitors inspired by the structure of thioridazine. Compounds were evaluated for their in vitro and ex vivo activity against <i>M. tuberculosis</i> H37Rv. Compared to the parent molecule, some of the compounds synthesized showed higher efflux inhibitory capacity, less cytotoxicity, and a remarkable synergistic effect with anti-TB drugs both in vitro and in human macrophages, demonstrating their potential to be used as coadjuvants for the treatment of tuberculosis

PRISMA flow diagram.

<p>PRISMA flow diagram.</p

Boosting Effect of 2‑Phenylquinoline Efflux Inhibitors in Combination with Macrolides against Mycobacterium smegmatis and Mycobacterium avium

The identification of efflux inhibitors to be used as adjuvants alongside existing drug regimens could have a tremendous value in the treatment of any mycobacterial infection. Here, we investigated the ability of four 2-(4′-propoxyphenyl)­quinoline Staphylococcus aureus NorA efflux inhibitors (<b>1</b>–<b>4</b>) to reduce the efflux activity in Mycobacterium smegmatis and Mycobacterium avium strains. All four compounds were able to inhibit efflux pumps in both mycobacterial species; in particular, <i>O</i>-ethylpiperazinyl derivative <b>2</b> showed an efflux inhibitory activity comparable to that of verapamil, the most potent mycobacterial efflux inhibitor reported to date, and was able to significantly reduce the MIC values of macrolides against different <i>M. avium</i> strains. The contribution of the <i>M. avium</i> efflux pumps MAV_1406 and MAV_1695 to clarithromycin resistance was proved because they were found to be overexpressed in two <i>M. avium</i> 104 isogenic strains showing high-level clarithromycin resistance. These results indicated a correlation between increased expression of efflux pumps, increased efflux, macrolide resistance, and reduction of resistance by efflux pump inhibitors such as compound <b>2</b>. Additionally, compound <b>2</b> showed synergistic activity with clarithromycin, at a concentration below the cytotoxicity threshold, in an ex vivo experiment against <i>M. avium</i> 104-infected macrophages. In summary, the 2-(4′-propoxyphenyl)­quinoline scaffold is suitable to obtain compounds endowed with good efflux pump inhibitory activity against both <i>S. aureus</i> and nontuberculous mycobacteria

PD catheters.

<p>Schematic diagram of the catheter segments including the silicone based segments: external <b>(a)</b> and intraperitoneal <b>(d)</b> and the cuffs: subcutaneous <b>(b)</b> and deep <b>(c)</b> (left). The different segments were cultured separately. Therefore, a Venn diagram (right) illustrates the possible relationships among them (external segment in red, subcutaneous cuff in yellow, deep cuff in blue and intraperitoneal segment in green) <b>(A)</b>. The representation of the distribution and overlap of positive segment cultures in catheters removed from patients with (left) and without infection (right) <b>(B)</b>. Values represent the number of catheters with a specific catheter colonization pattern. Four patients had growth on all catheter segments—with refractory and fungal (n = 3) and catheter-related peritonitis (n = 1); 2 patients had growth on the catheter subcutaneous and deep cuffs—with relapsing peritonitis (n = 1) and chronic catheter infection (n = 1); 3 patients had growth only on the catheter deep cuff—with relapsing peritonitis (n = 1) and chronic catheter infection (n = 2); 2 patients had growth on the catheter deep cuff and intraperitoneal segment—with chronic catheter infection; 3 patients had growth on the catheter subcutaneous and deep cuffs and intraperitoneal segment—with catheter-related peritonitis (n = 1), chronic catheter infection (n = 2). At the time of removal, ^§2 and *, 1 catheter did not present subcutaneous cuff.</p

Effect of PD solutions on <i>P. aeruginosa</i> and CNS biofilm cells (A) and biomass, cells and extracellular matrix (B).

<p>Mean log CFU/cm2 (A) and Abs570nm/cm2 (B) after 24 h exposure to PD solutions were used for the generation of the heat maps. The strains are indicated on the right (Pa, <i>P. aeruginosa</i>; Se, S. epidermidis, Scc, S. caprae/capitis; Sha, S. haemolyticus; Sho, S. hominis). Strains derived from catheters of patients with infection are indicated with an asterisk. Main strains clusters are identified as group 1 (G1) and group 2 (G2). The conventional and biocompatible (bicarbonate, bicarbonate/lactate, icodextrin) PD solutions are indicated on the bottom, as well as the growth controls: positive (C+, TSB culture medium) and negative (C-, saline). Dendrograms across the top and left of the heat map show the relationship between PD solutions and strains, respectively. Distance values are depicted by the gradient colour ranging from green (lowest value) to red (highest value).</p

Average quantification of the relative expression level,by RT-qPCR,of the genes that code for efflux pumps in <i>M. tuberculosis</i>exposed to isoniazid.

<p>Average quantification of the relative expression level,by RT-qPCR,of the genes that code for efflux pumps in <i>M. tuberculosis</i>exposed to isoniazid.</p

Effect of PD solutions on <i>P. aeruginosa</i> and CNS planktonic cells.

<p>Mean log CFU/mL after 24 h exposure to PD solutions was used for the generation of the heat map. The strains are indicated on the right (Pa, P. aeruginosa; Se, S. epidermidis, Scc, S. caprae/capitis; Sha, S. haemolyticus; Sho, S. hominis). Strains derived from catheters of patients with infection are indicated with an asterisk. Main strains clusters are identified as group 1 (G1) and group 2 (G2). The conventional and biocompatible (bicarbonate, bicarbonate/lactate, icodextrin) PD solutions are indicated on the bottom, as well as the growth controls: positive (C+, TSB culture medium) and negative (C-, saline). The initial inoculum concentration was of ~1 × 107 CFU/mL (7 log CFU/mL). Dendrograms across the top and left of the heat map show the relationship between PD solutions and strains, respectively. Distance values are depicted by the gradient colour ranging from green (lowest value) to red (highest value).</p

Average quantification of the relative expression level,by RT-qPCR,of the genes that code for efflux pumps in <i>M. tuberculosis</i>exposed to rifampicin.

<p>Average quantification of the relative expression level,by RT-qPCR,of the genes that code for efflux pumps in <i>M. tuberculosis</i>exposed to rifampicin.</p

Clinic and epidemiologic characteristics of the study population.

<p>Clinic and epidemiologic characteristics of the study population.</p

Antimycobacterial activity of the ion channel blockers against <i>M. tuberculosis</i>-infected macrophages.

<p> Effect of the inhibitors on the intracellular survival of <i>M</i>. <i>tuberculosis</i> (Mtb) within human monocyte-derived macrophages, three days post infection. Isoniazid (INH) was tested at 0.1 μg/ml, verapamil (VP), 10 μg/ml; thioridazine (TZ), 2.5 μg/ml; chlorpromazine (CPZ), 1.25 μg/ml; haloperidol (HAL), 1.25 μg/ml; flupenthixol (FPX), 1.25 μg/ml. The results are presented as a mean of the percentage of the survival ± SD. RIF, rifampicin; R, resistant; MDR, multidrug resistant; XDR, extensively drug resistant. The results were considered significant when *<i>P</i> < 0.05 and highly significant when **<i>P</i> < 0.01.</p