Novel molecule combinations and corresponding hybrids targeting artemisinin-resistant Plasmodium falciparum parasites.

Malaria is still considered as the major parasitic disease and the development of artemisinin resistance does not improve this alarming situation. Based on the recent identification of relevant malaria targets in the artemisinin resistance context, novel drug combinations were evaluated against artemisinin-sensitive and artemisinin-resistant Plasmodium falciparum parasites. Corresponding hybrid molecules were also synthesized and evaluated for comparison with combinations and individual pharmacophores (e.g. atovaquone, mefloquine or triclosan). Combinations and hybrids showed remarkable antimalarial activity (IC50 = 0.6 to 1.1 nM for the best compounds), strong selectivity, and didn't present any cross-resistance with artemisinin. Moreover, the combination triclosan + atovaquone showed high activity against artemisinin-resistant parasites at the quiescent stage but the corresponding hybrid lost this pharmacological property. This result is essential since only few molecules active against quiescent artemisinin-resistant parasites are reported. Our promising results highlight the potential of these combinations and paves the way for pharmacomodulation work on the best hybrids.

Inhibition of Mycobacterium tuberculosis InhA: Design, synthesis and evaluation of new di-triclosan derivatives.

Multi-drug resistant tuberculosis (MDR-TB) represents a growing problem for global healthcare systems. In addition to 1.3 million deaths in 2018, the World Health Organisation reported 484,000 new cases of MDR-TB. Isoniazid is a key anti-TB drug that inhibits InhA, a crucial enzyme in the cell wall biosynthesis pathway and identical in Mycobacterium tuberculosis and M. bovis. Isoniazid is a pro-drug which requires activation by the enzyme KatG, mutations in KatG prevent activation and confer INH-resistance. 'Direct inhibitors' of InhA are attractive as they would circumvent the main clinically observed resistance mechanisms. A library of new 1,5-triazoles, designed to mimic the structures of both triclosan molecules uniquely bound to InhA have been synthesised. The inhibitory activity of these compounds was evaluated using isolated enzyme assays with 2 (5-chloro-2-(4-(5-(((4-(4-chloro-2-hydroxyphenoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) exhibiting an IC50 of 5.6 µM. Whole-cell evaluation was also performed, with 11 (5-chloro-2-(4-(5-(((4-(cyclopropylmethoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) showing the greatest potency, with an MIC99 of 12.9 µM against M. bovis.

First triclosan-based macrocyclic inhibitors of InhA enzyme.

Two macrocyclic derivatives based on the triclosan frame were designed and synthesized as inhibitors of Mycobacterium tuberculosis InhA enzyme. One of the two molecules M02 displayed promising inhibitory activity against InhA enzyme with an IC50 of 4.7 µM. Molecular docking studies of these two compounds were performed and confirmed that M02 was more efficient as inhibitor of InhA activity. These molecules are the first macrocyclic direct inhibitors of InhA enzyme able to bind into the substrate pocket. Furthermore, these biaryl ether compounds exhibited antitubercular activities comparable to that of triclosan against M. tuberculosis H37Rv strain.

Ligand- and Structure-Based Approaches of Escherichia coli FabI Inhibition by Triclosan Derivatives: From Chemical Similarity to Protein Dynamics Influence.

Enoyl-acyl carrier protein reductase (FabI) is the limiting step to complete the elongation cycle in type II fatty acid synthase (FAS) systems and is a relevant target for antibacterial drugs. E. coli FabI has been employed as a model to develop new inhibitors against FAS, especially triclosan and diphenyl ether derivatives. Chemical similarity models (CSM) were used to understand which features were relevant for FabI inhibition. Exhaustive screening of different CSM parameter combinations featured chemical groups, such as the hydroxy group, as relevant to distinguish between active/decoy compounds. Those chemical features can interact with the catalytic Tyr156. Further molecular dynamics simulation of FabI revealed the ionization state as a relevant for ligand stability. Also, our models point the balance between potency and the occupancy of the hydrophobic pocket. This work discusses the strengths and weak points of each technique, highlighting the importance of complementarity among approaches to elucidate EcFabI inhibitor's binding mode and offers insights for future drug discovery.

Synthesis and antibacterial profiles of targeted triclosan derivatives.

There is an ongoing urgent need for new targeted antibacterial compounds with novel mechanisms of action for the treatment of infections caused by bacteria that are resistant to currently available materials. Since the expression of glycosidase enzymes within bacteria is unequally distributed, glycoside derivatives of antibacterial agents offer potential as targeted prodrugs for bacterial infections. Herein we report the synthesis and characterisation of four α-D-glycopyranosides and three ß-D-glycopyranosides of the broad antibacterial agent triclosan, in generally good synthetic yields, and with excellent purities. Each glycoside was analysed to determine its ability to inhibit the growth of a wide range of Gram-negative and Gram-positive organisms, including many of clinical significance. All of the triclosan glycosides that were synthesized demonstrated antibacterial activity against many of the organisms that were examined. For example, ß-galactoside (3a) and α-arabinoside (3c) had MIC values of 0.5 µg/ml for several strains of S. aureus and S. haemolyticus. The triclosan glycosides were also generally found to be more water soluble and much more selective than the underivatized triclosan, making them ideal both for the targeted inhibition of bacterial growth and as agents for the selective recovery of bacteria from mixed cultures. In the latter case, two Bacillus strains could be identified from various strains of Bacillus and Staphylococcus after inoculation onto Nutrient Agar No. 2 with 0.25 µg/ml triclosan-α-D-glucopyranoside (3e). This glucoside may, therefore, be of use for the isolation and identification of the food-poisoning organism Bacillus cereus.

Triclosan-caffeic acid hybrids: Synthesis, leishmanicidal, trypanocidal and cytotoxic activities.

The synthesis, cytotoxicity, anti-leishmanial and anti-trypanosomal activities of twelve triclosan-caffeic acid hybrids are described herein. The structure of the synthesized products was elucidated by a combination of spectrometric analyses. The synthesized compounds were evaluated against amastigotes forms of L. (V) panamensis, which is the most prevalent Leishmania species in Colombia, and against Trypanosoma cruzi, which is the pathogenic species to humans. Cytotoxicity was evaluated against human U-937 macrophages. Eight compounds were active against L. (V) panamensis (18-23, 26 and 30) and eight of them against T. cruzi (19-22, 24 and 28-30) with EC50 values lower than 40 µM. Compounds 19-22, 24 and 28-30 showed higher activities than benznidazole (BNZ). Esters 19 and 21 were the most active compounds for both L. (V) panamensis and T. cruzi with 3.82 and 11.65 µM and 8.25 and 8.69 µM, respectively. Compounds 19-22, 24 and 28-30 showed higher activities than benznidazole (BNZ). Most of the compounds showed antiprotozoal activity and with exception of 18, 26 and 28, the remaining compounds were toxic for mammalian cells, yet they have potential to be considered as candidates for anti-trypanosomal and anti-leishmanial drug development. The activity is dependent on the length of the alkyl linker with compound 19, bearing a four-carbon alkyl chain, the most performing hybrid. In general, hydroxyl groups increase both activity and cytotoxicity and the presence of the double bond in the side chain is not decisive for cytotoxicity and anti-protozoal activity.

Synthesis, leishmanicidal and cytotoxic activity of triclosan-chalcone, triclosan-chromone and triclosan-coumarin hybrids.

Twelve hybrids derived from triclosan were obtained via Williamson etherification of O-triclosan alkyl bromide plus chalcone and O-coumarin or O-chromone alkyl bromide plus triclosan, respectively. Structures of the products were elucidated by spectroscopic analysis. The synthesized compounds were evaluated for antileishmanial activity against L. (V) panamensis amastigotes. Cytotoxic activity was also evaluated against mammalian U-937 cells. Compounds 7-9 and 17, were active against Leishmania parasites (EC50=9.4; 10.2; 13.5 and 27.5 µg/mL, respectively) and showed no toxicity toward mammalian cells (>200 µg/mL). They are potential candidates for antileishmanial drug development. Compounds 25-27, were active and cytotoxic. Further studies using other cell types are needed in order to discriminate whether the toxicity shown by these compounds is against tumor or non-tumor cells. The results indicate that compounds containing small alkyl chains show better selectivity indices. Moreover, Michael acceptor moieties may modify both the leishmanicidal activity and cytotoxicity. Further studies are required to evaluate if the in vitro activity against Leishmania panamensis demonstrated here is also observed in vivo.

Modification of triclosan scaffold in search of improved inhibitors for enoyl-acyl carrier protein (ACP) reductase in Toxoplasma gondii.

Through our focused effort to discover new and effective agents against toxoplasmosis, a structure-based drug design approach was used to develop a series of potent inhibitors of the enoyl-acyl carrier protein (ACP) reductase (ENR) enzyme in Toxoplasma gondii (TgENR). Modifications to positions 5 and 4' of the well-known ENR inhibitor triclosan afforded a series of 29 new analogues. Among the resulting compounds, many showed high potency and improved physicochemical properties in comparison with the lead. The most potent compounds 16 a and 16 c have IC50 values of 250 nM against Toxoplasma gondii tachyzoites without apparent toxicity to the host cells. Their IC50 values against recombinant TgENR were found to be 43 and 26 nM, respectively. Additionally, 11 other analogues in this series had IC50 values ranging from 17 to 130 nM in the enzyme-based assay. With respect to their excellent in vitro activity as well as improved drug-like properties, the lead compounds 16 a and 16 c are deemed to be excellent starting points for the development of new medicines to effectively treat Toxoplasma gondii infections.

Development of a triclosan scaffold which allows for adaptations on both the A- and B-ring for transport peptides.

The enoyl acyl-carrier protein reductase (ENR) enzyme is harbored within the apicoplast of apicomplexan parasites providing a significant challenge for drug delivery, which may be overcome through the addition of transductive peptides, which facilitates crossing the apicoplast membranes. The binding site of triclosan, a potent ENR inhibitor, is occluded from the solvent making the attachment of these linkers challenging. Herein, we have produced 3 new triclosan analogs with bulky A- and B-ring motifs, which protrude into the solvent allowing for the future attachment of molecular transporters for delivery.

Design, synthesis, and biological activity of diaryl ether inhibitors of Toxoplasma gondii enoyl reductase.

Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme for parasite survival. In view of triclosan's poor druggability, which limits its therapeutic use, a new set of B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display improved solubility, permeability and a comparable MIC50 value to that of triclosan. Modeling of these inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the B-ring modifications have additional interactions with the strongly conserved Asn130.

From triclosan toward the clinic: discovery of nonbiocidal, potent FabI inhibitors for the treatment of resistant bacteria.

In this paper, we present some elements of our optimization program to decouple triclosan's specific FabI effect from its nonspecific cytotoxic component. The implementation of this strategy delivered highly specific, potent, and nonbiocidal new FabI inhibitors. We also disclose some preclinical data of one of their representatives, 83, a novel antibacterial compound active against resistant staphylococci and some clinically relevant Gram negative bacteria that is currently undergoing clinical trials.

Novel, UV-curable coatings containing a tethered biocide: Synthesis, characterization, and antimicrobial activity.

Cationic, UV -curable coatings containing the tethered biocide, triclosan, were produced and their antimicrobial activity toward Staphylococcus epidermidis and Escherichia coli determined. Two polysiloxanes functionalized with both cycloaliphatic epoxy and triclosan were synthesized using hydrosilylation. The functionalized polysiloxanes, with varied concentration of pendant triclosan, were used to produce UV-curable coatings with reasonably good coating properties. Fourier transform (FT)-Raman spectroscopy showed that the tethered triclosan moieties self-concentrate on the coating surface. Using biological assays, it was determined that the coatings possessed nearly 100% antimicrobial activity toward the Gram-positive bacterium, S. epidermidis, without leaching toxic components. For the Gram-negative bacterium, E. coli, 60-80% reduction in biofilm retention was observed for all the coatings. Interestingly, the coatings were lesser effective in reducing E. coli cell viability suggesting that the tethered triclosan were able to substantially reduce the production of the biofilm extracellular matrix with minimal adverse affect on the bacterial cells attached to the coating surfaces. The high specificity of the coatings toward S. epidermidis indicates that a novel mode of contact-active antimicrobial activity was achieved through the disruption of processes unique to the Gram-positive cell wall. These novel UV-curable coatings have potential applications in inhibiting implantable biomedical device associated infections.

Design, development, synthesis, and docking analysis of 2'-substituted triclosan analogs as inhibitors for Plasmodium falciparum enoyl-ACP reductase.

A structure-based approach has been adopted to develop 2'-substituted analogs of triclosan. The Cl at position 2' in ring B of triclosan was chemically substituted with other functional groups like NH(2), NO(2) and their inhibitory potencies against PfENR were determined. The binding energies of the 2' substituted analogs of triclosan for enoyl-acyl carrier protein reductase (ENR) of Plasmodium falciparum were determined using Autodock. Based on the autodock results, we synthesized the potential compounds. The IC(50) and inhibition constant (K(i)) of 2' substituted analogs of triclosan were determined against purified PfENR. Among them, two compounds, 2-(2'-Amino-4'-chloro-phenoxy)-5-chloro-phenol (compound 4) and 5-chloro-2-(4'-chloro-2'-nitro-phenoxy)-phenol) (compound 5) exhibited good potencies. Compound 4 followed uncompetitive inhibition kinetics with crotonoyl CoA and competitive with NADH. It was shown to have an IC(50) of 110 nM; inhibition constant was 104 nM with the substrate and 61 nM with the cofactor. IC(50) of compound 5 was determined to be 229 nM. Compounds 4 and 5 showed significant inhibition of the parasite growth in P. falciparum culture.

Design, synthesis, and application of novel triclosan prodrugs as potential antimalarial and antibacterial agents.

A number of new triclosan-conjugated analogs bearing biodegradable ester linkage have been synthesized, characterized and evaluated for their antimalarial and antibacterial activities. Many of these compounds exhibit good inhibition against Plasmodium falciparum and Escherichia coli. Among them tertiary amine containing triclosan-conjugated prodrug (5) inhibited both P. falciparum (IC(50); 0.62microM) and E. coli (IC(50); 0.26microM) at lower concentrations as compared to triclosan. Owing to the presence of a cleavable ester moiety, these new prodrugs are hydrolyzed under physiological conditions and parent molecule, triclosan, is released. Further, introduction of tertiary/quaternary functionality increases their cellular uptake. These properties impart them with higher potency to their antimalarial as well as antibacterial activities. The best compound among them 5 shows close to four-fold enhanced activities against P. falciparum and E. coli cultures as compared to triclosan.

Preparation and characterization of triclosan nanoparticles for periodontal treatment.

The aim of this work was to produce and characterize triclosan-loaded nanoparticles (NPs) by the emulsification-diffusion process, in an attempt to obtain a novel delivery system adequate for the treatment of periodontal disease. The NPs were prepared using poly(D,L-lactide-co-glycolide) (PLGA), poly(D,L-lactide) (PLA) and cellulose acetate phthalate (CAP). Poly(vinyl alcohol) (PVAL) was used as stabilizer. Batches were prepared with different amounts of triclosan (TCS) in order to evaluate the influence of drug on NP properties. Solid NPs of less than 500 nm in diameter were obtained. Entrapment efficiencies were higher than 63.8%. The characterization by scanning electron microscopy and light scattering indicated that high concentrations of TCS seemingly caused the increase of NP mean size. A decrease in the PLGA glass transition temperature was observed by differential scanning calorimetry. This could indicate that TCS in PLGA-NPs behaves as a non-conventional plasticizer. Subsequently, in vitro release studies were carried out under sink conditions using a device designed in our laboratory to allow a direct contact between the particles and the dissolution medium. A fast release of TCS from NPs was detected. A preliminary in vivo study in dogs with induced periodontal defects suggested that TCS-loaded NPs penetrate through the junctional epithelium.

Incorporation of low molecular weight biocides into polystyrene-divinyl benzene beads with controlled release characteristics.

Triclosan and phosphonium salt biocides have been separately incorporated into polystyrene-divinylbenzene (PS-DVB) beads by suspension polymerization. Ultraviolet (UV) absorption measurements have been used to monitor the release of these low molecular weight biocides out of the PS-DVB beads immersed in water-ethanol mixtures and in physiological saline. The release of the biocide agents is strongly dependent on either the DVB or/and the antimicrobial composition ratio in the beads. An increase of biocide incorporation in the PS/DVB beads was accompanied by a corresponding enhancement of its concentration in liquid mixtures. On the contrary, higher cross-linking densities hindered the biocide migration out of the beads by diminishing its release rate into either the aqueous ethanol solutions or the natural serum. Moreover, Fourier transform Raman (FT-Raman) spectra and Attenuated Total Reflectance Infrared (ATR-FTIR) measurements of the PS-DVB-Triclosan and PS-DVB-phosphonium salt beads, before and after their immersion in water-ethanol solutions, gave a similar qualitative evidence of the biocide release.

Inhibition of the bacterial enoyl reductase FabI by triclosan: a structure-reactivity analysis of FabI inhibition by triclosan analogues.

To explore the molecular basis for the picomolar affinity of triclosan for FabI, the enoyl reductase enzyme from the type II fatty acid biosynthesis pathway in Escherichia coli, an SAR study has been conducted using a series of triclosan analogues. Triclosan (1) is a slow, tight-binding inhibitor of FabI, interacting specifically with the E.NAD(+) form of the enzyme with a K(1) value of 7 pM. In contrast, 2-phenoxyphenol (2) binds with equal affinity to the E.NAD(+) (K(1) = 0.5 microM) and E.NADH (K(2) = 0.4 microM) forms of the enzyme and lacks the slow-binding step observed for triclosan. Thus, removal of the three triclosan chlorine atoms reduces the affinity of the inhibitor for FabI by 70,000-fold and removes the preference for the E.NAD(+) FabI complex. 5-Chloro-2-phenoxyphenol (3) is a slow, tight-binding inhibitor of FabI and binds to the E.NAD(+) form of the enzyme (K(1) = 1.1 pM) 7-fold more tightly than triclosan. Thus, while the two ring B chlorine atoms are not required for FabI inhibition, replacement of the ring A chlorine increases binding affinity by 450,000-fold. Given this remarkable observation, the SAR study was extended to the 5-fluoro-2-phenoxyphenol (4) and 5-methyl-2-phenoxyphenol (5) analogues to further explore the role of the ring A substituent. While both 4 and 5 are slow, tight-binding inhibitors, they bind substantially less tightly to FabI than triclosan. Compound 4 binds to both E.NAD(+) and E.NADH forms of the enzyme with K(1) and K(2) values of 3.2 and 240 nM, respectively, whereas compound 5 binds exclusively to the E.NADH enzyme complex with a K(2) value of 7.2 nM. Thus, the ring A substituent is absolutely required for slow, tight-binding inhibition. In addition, pK(a) measurements coupled with simple electrostatic calculations suggest that the interaction of the ring A substituent with F203 is a major factor in governing the affinity of analogues 3-5 for the FabI complex containing the oxidized form of the cofactor.