Triclosan Analogues Research Articles

Application of Machine Learning in the Development of Fourth Degree Quantitative Structure–Activity Relationship Model for Triclosan Analogs Tested against Plasmodium falciparum 3D7

Application of Machine Learning in the Development of Fourth Degree Quantitative Structure–Activity Relationship Model for Triclosan Analogs Tested against <i>Plasmodium falciparum</i> 3D7

SYNTHESIS, CHARACTERIZATION, AND IN VITRO ANTIMALARIAL ACTIVITY OF DIHYDROXYLATION DERIVATIVES OF TRICLOSAN

Objective: The emergence of malaria as a global health problem over the past few decades, accompanied by the rise of chemoresistant strains ofPlasmodium falciparum, has emphasized the need for the discovery of new therapeutic drugs against this disease. In this study, enantiomericallyenriched (enantioenriched) analogs of triclosan were synthesized and evaluated for antimalarial activity against P. falciparum cultures.Methods: Enantioselective dihydroxylation of the olefin in amide seven was performed efficiently using chiral quinine ligand (DHQ)2PHAL to yieldenantioenriched dihydroxy propionamide derivative (+)-1 in moderate yields. In a similar way, the chiral quinidine ligand (DHQD)2PHAL was used asstereoselectivity agent yielded the desired enantioenriched (−)-1. The enantioenriched products were used for further in vitro assay, and accordingly thepercent enantiomeric excess (% ee) was not determined. The structures of compounds were proven by spectral data (1H NMR, 13C NMR, and mass spectra).Results: The phenol moiety at the C1 position of triclosan was chemically substituted with a methoxy group, in conjunction with an introducedstereocenter in a 2,3-dihydroxy-propionamide group at C2’ position. Unmodified triclosan inhibited the P. falciparum cultures with an IC50 value of27.2 μM. By contrast, the triclosan analogs, compounds (+)-1 and (−)-1, inhibited the P. falciparum cultures with IC50 values of 0.034 and 0.028 μM,respectively.Conclusion: Collectively, our preliminary in vitro results suggest that these triclosan analogs have potent antimalarial activity and represent apromising new treatment strategy on further development.

An Efficient and Economical Assay to Screen for Triclosan Binding to FabI

Triclosan is an effective inhibitor for enoyl acyl carrier protein reductase (ENR) in fatty acid biosynthesis. Triclosan-resistant mutants of ENR have emerged. Thus, it is important to detect these triclosan-resistant mutations in ENR. Generally, enzyme activity assays on the mutants are used to determine the effect of triclosan on ENR activity. Since the substrates are linked to acyl carrier protein (ACP), the assays are challenging due to the need to prepare the ACP and link it to the substrates. Non-ACP-linked (coenzyme A [CoA]-linked) substrates can be used in some ENR, but not in all. Consequently, screening for triclosan-resistant mutants is also challenging. We have developed a simple thermal shift assay, which does not use ACP-linked substrates, to determine the binding ability of triclosan to the ENR active site, and thus it can be used for screening for triclosan-resistant mutants. Staphylococcus aureus FabI enzyme and its mutants were used to demonstrate the binding ability of triclosan with NADP+ to FabI. The direct correlation between the binding ability and enzyme activity was demonstrated with Francisella tularensis FabI. This method may also be applied to select effective triclosan analogues that inhibit ENR activity.

Design, synthesis and evaluation of antitubercular activity of Triclosan analogues

Novel Triclosan mimic diphenyl ether derivatives 4a–k were designed and synthesized with lipophilicity considerably lesser than that of Triclosan. The binding mode of the compounds at the active site of enoyl-ACP reductase was analysed using docking method. The syntheses were carried out with one-pot reductive amination reaction and were characterized by spectral techniques. The synthesized compounds were evaluated for their in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv strain by Microplate Alamar Blue assay. Compounds 4h and 4j were the most active compounds with MIC equal to 25μg/mL against M. tuberculosis H37Rv strain. All compounds were also examined for their cytotoxic potential against VERO and HepG2 cell lines and were safe even at 300μg/mL. LogP of all the synthesized compounds was evaluated by RPHPLC and was significantly lesser than Triclosan.

Discrimination of Potent Inhibitors of Toxoplasma gondii Enoyl-Acyl Carrier Protein Reductase by a Thermal Shift Assay

Many microbial pathogens rely on a type II fatty acid synthesis (FASII) pathway that is distinct from the type I pathway found in humans. Enoyl-acyl carrier protein reductase (ENR) is an essential FASII pathway enzyme and the target of a number of antimicrobial drug discovery efforts. The biocide triclosan is established as a potent inhibitor of ENR and has been the starting point for medicinal chemistry studies. We evaluated a series of triclosan analogues for their ability to inhibit the growth of Toxoplasma gondii, a pervasive human pathogen, and its ENR enzyme (TgENR). Several compounds that inhibited TgENR at low nanomolar concentrations were identified but could not be further differentiated because of the limited dynamic range of the TgENR activity assay. Thus, we adapted a thermal shift assay (TSA) to directly measure the dissociation constant (Kd) of the most potent inhibitors identified in this study as well as inhibitors from previous studies. Furthermore, the TSA allowed us to determine the mode of action of these compounds in the presence of the reduced nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide (NAD⁺) cofactor. We found that all of the inhibitors bind to a TgENR-NAD⁺ complex but that they differed in their dependence on NAD⁺ concentration. Ultimately, we were able to identify compounds that bind to the TgENR-NAD⁺ complex in the low femtomolar range. This shows how TSA data combined with enzyme inhibition, parasite growth inhibition data, and ADMET predictions allow for better discrimination between potent ENR inhibitors for the future development of medicine.

Design, Synthesis, and Biological and Crystallographic Evaluation of Novel Inhibitors of Plasmodium falciparum Enoyl-ACP-reductase (PfFabI)

Malaria, a disease of worldwide significance, is responsible for over one million deaths annually. The liver-stage of Plasmodium's life cycle is the first, obligatory, but clinically silent step in malaria infection. The P. falciparum type II fatty acid biosynthesis pathway (PfFAS-II) has been found to be essential for complete liver-stage development and has been regarded as a potential antimalarial target for the development of drugs for malaria prophylaxis and liver-stage eradication. In this paper, new coumarin-based triclosan analogues are reported and their biological profile is explored in terms of inhibitory potency against enzymes of the PfFAS-II pathway. Among the tested compounds, 7 and 8 showed the highest inhibitory potency against Pf enoyl-ACP-reductase (PfFabI), followed by 15 and 3. Finally, we determined the crystal structures of compounds 7 and 11 in complex with PfFabI to identify their mode of binding and to confirm outcomes of docking simulations.

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.

Novel diaryl ureas with efficacy in a mouse model of malaria

Exploration of triclosan analogs has led to novel diaryl ureas with significant potency against in vitro cultures of drug-resistant and drug-sensitive strains of the human malaria parasite Plasmodium falciparum. Compound 18 demonstrated EC50 values of 37 and 55nM versus in vitro cultured parasite strains and promising in vivo efficacy in a Plasmodium berghei antimalarial mouse model, with >50% survival at day 31 post-treatment when administered subcutaneously at 256mg/kg. This series of compounds provides a chemical scaffold of novel architecture, as validated by cheminformatics analysis, to pursue antimalarial drug discovery efforts.

Design, development, synthesis, and docking analysis of 2′-substituted triclosan analogs as inhibitors forPlasmodium falciparumEnoyl-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.

Triclosan derivatives: towards potent inhibitors of drug-sensitive and drug-resistant Mycobacterium tuberculosis.

Triclosan has been previously shown to inhibit InhA, an essential enoyl acyl carrier protein reductase involved in mycolic acid biosynthesis, the inhibition of which leads to the lysis of Mycobacterium tuberculosis. Using a structure-based drug design approach, a series of 5-substituted triclosan derivatives was developed. Two groups of derivatives with alkyl and aryl substituents, respectively, were identified with dramatically enhanced potency against purified InhA. The most efficacious inhibitor displayed an IC(50) value of 21 nM, which was 50-fold more potent than triclosan. X-ray crystal structures of InhA in complex with four triclosan derivatives revealed the structural basis for the inhibitory activity. Six selected triclosan derivatives were tested against isoniazid-sensitive and resistant strains of M. tuberculosis. Among those, the best inhibitor had an MIC value of 4.7 microg mL(-1) (13 microM), which represents a tenfold improvement over the bacteriocidal activity of triclosan. A subset of these triclosan analogues was more potent than isoniazid against two isoniazid-resistant M. tuberculosis strains, demonstrating the significant potential for structure-based design in the development of next generation antitubercular drugs.

Design and in silico screening of combinatorial library of antimalarial analogs of triclosan inhibiting Plasmodium falciparum enoyl-acyl carrier protein reductase

Enoyl-acyl carrier protein reductase of Plasmodium falciparum (PfENR) is an important target for antimalarial agents that interfere with the FAS-II pathway of lipid synthesis, which is specific for the parasite. Recent studies showed that substituted analogs of triclosan (TCL) inhibit the purified PfENR enzyme with IC 50 values below 200 nM when the suboptimal 5-chloro group was replaced by larger hydrophobic moieties. We have used computer-assisted combinatorial techniques to design, focus and in silico screen a virtual library of TCL analogs substituted at positions 5, 4′ and 2′. Our study can thus direct synthetic chemists working on the antimalarial FAS-II inhibitors towards the explored subset of the chemical space, which is predicted to contain compounds with PfENR inhibition potencies in the low nanomolar range and favorable ADME properties.

The Fatty Acid Biosynthesis Enzyme FabI Plays a Key Role in the Development of Liver-Stage Malarial Parasites

The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.

X-ray Structural Analysis of Plasmodium falciparum Enoyl Acyl Carrier Protein Reductase as a Pathway toward the Optimization of Triclosan Antimalarial Efficacy

The x-ray crystal structures of five triclosan analogs, in addition to that of the isoniazid-NAD adduct, are described in relation to their integral role in the design of potent inhibitors of the malarial enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of the novel 5-substituted analogs exhibit low micromolar potency against in vitro cultures of drug-resistant and drug-sensitive strains of the P. falciparum parasite and inhibit purified PfENR enzyme with IC50 values of <200 nM. This study has significantly expanded the knowledge base with regard to the structure-activity relationship of triclosan while affording gains against cultured parasites and purified PfENR enzyme. In contrast to a recent report in the literature, these results demonstrate the ability to improve the in vitro potency of triclosan significantly by replacing the suboptimal 5-chloro group with larger hydrophobic moieties. The biological and x-ray crystallographic data thus demonstrate the flexibility of the active site and point to future rounds of optimization to improve compound potency against purified enzyme and intracellular Plasmodium parasites.

Insight through Molecular Mechanics Poisson−Boltzmann Surface Area Calculations into the Binding Affinity of Triclosan and Three Analogues for FabI, the E. coli Enoyl Reductase

Keeping pace with emerging drug resistance in clinically important pathogens will be greatly aided by inexpensive yet reliable computational methods that predict the binding affinities of ligands for drug targets. We present results using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method to calculate the affinity of a series of triclosan analogues for the E. coli enoyl reductase FabI, spanning a 450000-fold range of binding affinities. Significantly, a high correlation is observed between the calculated binding energies and those determined experimentally. Further examination indicates that the van der Waals energies are the most correlated component of the total affinity (r2 = 0.74), indicating that the shape of the inhibitor is very important in defining the binding energies for this system. The validation of MM-PBSA for the E coli FabI system serves as a platform for inhibitor design efforts focused on the homologous enzyme in Staphylococcus aureus and Mycobacterium tuberculosis.

Synthesis and biological activity of diaryl ether inhibitors of malarial enoyl acyl carrier protein reductase. Part 2: 2′-Substituted triclosan derivatives

2′-Substituted analogs of triclosan have been synthesized to target inhibition of the key malarial enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of these compounds exhibit good potency (EC 50 < 500 nM) against in vitro cultures of drug-resistant and drug-sensitive strains of the P. falciparum parasite and modest (IC 50 = 1–20 μM) potency against purified PfENR enzyme. Compared to triclosan, this survey of 2′-substituted derivatives has afforded gains in excess of 20- and 30-fold versus the 3D7 and Dd2 strains of parasite, respectively.

Synthesis, biological activity, and X-ray crystal structural analysis of diaryl ether inhibitors of malarial enoyl acyl carrier protein reductase. Part 1: 4′-Substituted triclosan derivatives

A structure-based approach has been taken to develop 4′-substituted analogs of triclosan that target the key malarial enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of these compounds exhibit nanomolar potency against purified PfENR enzyme and modest (2–10 μM) potency against in vitro cultures of drug-resistant and drug-sensitive strains of the P. falciparum parasite. X-ray crystal structures of nitro 29, aniline 30, methylamide 37, and urea 46 demonstrate the presence of hydrogen-bonding interactions with residues in the active site and point to future rounds of optimization to improve compound potency against purified enzyme and intracellular parasites.

Identification of Inhibitors of Bacterial Enoyl-Acyl Carrier Protein Reductase

The FabI-related enoyl-ACP reductase enzymes of bacteria meet many of the criteria for antibacterial targets. These enzymes are essential for the growth of several pathogenic species, have no significant mammalian homologs, catalyze a rate-limiting step in a vital macromolecular biosynthetic pathway, and are already the targets of antibacterials used in the clinic (isoniazid) and in consumer products (triclosan). The suitability of FabI as an antibiotic target is diminished somewhat by the discovery that many pathogens carry an alternate unrelated enoyl-ACP reductase (FabK) or both reductases. However, a key human pathogen, Staphylococcus aureus and its increasingly common drug-resistant derivative MRSA are sensitive to FabI inhibitors. Screening for inhibitors of this target has resulted in the identification of five chemical classes of potent inhibitors. In addition, analogs of triclosan with increased potency and with pro-drug features have been engineered. At least one of these classes of inhibitors has been optimized and tested in animals for pharmacokinetic properties and efficacy. Further development of one or more of these classes and further screening are expected to generate new FabI inhibitors for application in the clinic against drug-resistant S. aureus.

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.

Structural Elucidation of the Specificity of the Antibacterial Agent Triclosan for Malarial Enoyl Acyl Carrier Protein Reductase

The human malaria parasite Plasmodium falciparum synthesizes fatty acids using a type II pathway that is absent in humans. The final step in fatty acid elongation is catalyzed by enoyl acyl carrier protein reductase, a validated antimicrobial drug target. Here, we report the cloning and expression of the P. falciparum enoyl acyl carrier protein reductase gene, which encodes a 50-kDa protein (PfENR) predicted to target to the unique parasite apicoplast. Purified PfENR was crystallized, and its structure resolved as a binary complex with NADH, a ternary complex with triclosan and NAD(+), and as ternary complexes bound to the triclosan analogs 1 and 2 with NADH. Novel structural features were identified in the PfENR binding loop region that most closely resembled bacterial homologs; elsewhere the protein was similar to ENR from the plant Brassica napus (root mean square for Calphas, 0.30 A). Triclosan and its analogs 1 and 2 killed multidrug-resistant strains of intra-erythrocytic P. falciparum parasites at sub to low micromolar concentrations in vitro. These data define the structural basis of triclosan binding to PfENR and will facilitate structure-based optimization of PfENR inhibitors.