In particular the B-ring is exposed to the outside solvent via a channel which would allow the fatty acyl substrate attached to the acyl carrier protein to enter the active site (Fig. Infection acquired during pregnancy can be especially severe; it can transmit from mother to the fetus, leading to congenital toxoplasmosis which may result in abortion, neonatal death, or fetal abnormalities.2, 10C16 Currently, there is no vaccine available to prevent human infection from this pathogen. Antifolate agents, sulfadiazine and pyrimethamine, are two primary medicines for treatment of infection in humans.2, 15 Although these medicines are effective against tachyzoites in the acute stage of the disease, they do not eradicate encysted, latent bradyzoites. Furthermore, these therapies can be associated with side effects such as bone marrow depression, hypersensitivity and skin rashes.15, 16 There is an urgent need to develop new anti-medicines that are both efficacious and nontoxic to humans. One attractive target for chemotherapeutic intervention against apicomplexan parasites is the prokaryotic-like type II fatty acid biosynthesis (FAS-II) pathway.17C21 In and studies.21 Type II FAS is fundamentally divergent from the analogous FAS I pathway in mammals. In eukaryotes, fatty acid biosynthetic enzymes integrate on a single multifunctional polypeptide 3-Methyl-2-oxovaleric acid (FASI), whereas fatty acid synthesis in prokaryotes utilizes a set of distinct enzymes composing the FAS-II pathway.22 Fatty acid biosynthesis is an iterative process beginning with condensation of acetyl-Coenzyme A 3-Methyl-2-oxovaleric acid (acetyl-CoA) with a growing fatty acid chain. In parasites with an IC50 value of ~200nM.18 Although triclosan is a potent inhibitor of TgENR, the diphenyl ether has low water solubility and a high ClogP value. Another major challenge for the development of medicines against targets which reside within the apicoplast of apicomplexan parasites is the need for the inhibitors to cross the four membranes of the parasite-specific organelle, in addition to the barriers set by both host cell and the parasite.27 In order to overcome these structural drawbacks and address the uptake problem, structure-based modification of triclosan was directed by improving the ADMET (absorption, distribution, metabolism, excretion and toxicity) profiles with special focus on the increase of aqueous solubility and permeability. We have previously shown that the A-ring of triclosan can be modified to exploit an additional space at the base of the inhibitor binding pocket.28 Furthermore, we have shown that substitution on the B-ring may also be tolerated to produce effective triclosan analogues {Stec et al., and using methods that have been described in the recent literature28, 33 For comparison of parasite burden between treatment groups, analysis of variance (ANOVA) was performed with group and run as factors. Due to evidence of non-normality, natural log-transformed parasite burden was used in the analysis.(Also, see Supplementary Material). The crystal structure of TgENR in complex with NAD+ and triclosan,34 showed that the 4-chloro phenoxy ring (A-ring) of triclosan participates in a – stacking interaction with NAD+, and a hydrogen bond forms between the hydroxyl group and Tyr189. However, the 2,4-dichlorophenoxy ring (B-ring) engages only in van der Waals interactions within a pocket encompassed by the peptide backbone of residues Leu128 to Ala131, the pyrophosphate and nicotinamide moieties of NAD+, and the side chains of Val134, Met193, Ala231 and Ile235. Moreover, there remains some additional space around the B-ring that could be exploited. In particular the B-ring is exposed to the outside solvent via a channel which would allow the fatty acyl substrate attached to the acyl carrier protein to enter the active site (Fig. 1A). Open in a separate window Figure 1 (A) The TgENR/NAD+/triclosan crystal structure shown in a cartoon representation covered by a transparent surface showing the channel which leads from the triclosan inhibitor to the outside solvent. TgENR Asn130 which has been targeted for the design of new inhibitors is shown in stick format close to the B-ring of triclosan. (B) A multiple sequence alignment in the vicinity of TgENR Asn130 (shown by an asterisk) of a number of parasitic and plant ENR enzymes showing full sequence conservation across parasite and plant families. In view of the space around the B-ring which could be.A lower dose of 50mg/kg was not effective and a higher dose of 100mg/kg was not completely soluble. pathogen. Antifolate agents, sulfadiazine and pyrimethamine, are two primary medicines for treatment of infection in humans.2, 15 Although these medicines are effective against tachyzoites in the acute stage of the disease, they do not eradicate encysted, latent 3-Methyl-2-oxovaleric acid bradyzoites. Furthermore, these therapies can be associated with side effects such as bone marrow depression, hypersensitivity and skin rashes.15, 16 There is an urgent need to develop new anti-medicines that are both efficacious and nontoxic to humans. One attractive target for chemotherapeutic intervention against apicomplexan parasites is the prokaryotic-like type II fatty acid biosynthesis (FAS-II) pathway.17C21 In and studies.21 Type II FAS is fundamentally divergent from the analogous FAS I pathway in mammals. In eukaryotes, fatty acid biosynthetic enzymes integrate on a single multifunctional polypeptide (FASI), whereas fatty acid synthesis in prokaryotes utilizes a set of distinct enzymes composing the FAS-II pathway.22 Fatty acid biosynthesis is an iterative process beginning with condensation of acetyl-Coenzyme A (acetyl-CoA) with a growing fatty acid chain. In parasites with an IC50 value of ~200nM.18 Although triclosan is a potent inhibitor of TgENR, the diphenyl ether has low water solubility and a high ClogP value. Another major challenge for the development of medicines against targets which reside within the apicoplast of apicomplexan parasites is the need for the inhibitors to cross the four membranes of the parasite-specific organelle, in addition to the barriers set by both host cell and the parasite.27 In order to overcome these structural drawbacks and address the uptake problem, structure-based modification of triclosan was directed by improving the ADMET (absorption, distribution, metabolism, excretion and toxicity) profiles with special focus on the increase of aqueous solubility and permeability. We have previously shown that the A-ring of triclosan can be modified to exploit an additional space at the base of the inhibitor binding pocket.28 Furthermore, we have shown that substitution on the B-ring may also be tolerated to produce effective triclosan analogues {Stec et al., and using methods that have been described in the recent literature28, 33 For comparison of parasite burden between treatment groups, analysis of variance (ANOVA) was performed with group and run as factors. Due to evidence of non-normality, natural log-transformed parasite burden was used in the analysis.(Also, see Supplementary Material). The crystal structure of TgENR in complex with NAD+ and triclosan,34 showed that the 4-chloro phenoxy ring (A-ring) of triclosan participates in a – stacking interaction with NAD+, and a hydrogen bond forms between the hydroxyl group and Tyr189. However, the 2,4-dichlorophenoxy ring (B-ring) engages only in van der Waals interactions within a pocket encompassed by the peptide backbone of residues Leu128 to Ala131, the pyrophosphate and nicotinamide moieties of NAD+, and the side chains of Val134, Met193, Ala231 and Ile235. Moreover, there remains some additional space around the B-ring that could be exploited. In particular the B-ring is exposed to the outside solvent via a channel which would allow the fatty acyl substrate attached to the acyl carrier protein to enter the active site (Fig. 1A). Open in a separate window Figure 1 (A) The TgENR/NAD+/triclosan crystal structure shown in a cartoon representation covered by a transparent surface showing the channel which leads from the triclosan inhibitor to the outside solvent. TgENR Asn130 which has been targeted for the design 3-Methyl-2-oxovaleric acid of new inhibitors is shown in stick format close to the B-ring of triclosan. (B) A multiple sequence alignment in the vicinity of TgENR Asn130 (shown by an asterisk) of a number of parasitic and plant ENR enzymes showing full sequence conservation across parasite and plant families. In view of the space around the B-ring which could be exploited, we devised a modification strategy to change the B- ring, incorporating additional polar groups to optimize the physicochemical properties (such as permeability and solubility) while keeping the A-ring intact. These modifications were guided by computer-aided ADMET prediction (ADMET suite from ACD/labs). In total, 19 triclosan analogs have been designed with either substitutions at the 4′-position of the B-ring or with the complete replacement of its benzene ring by heteroaromatic groups. The biological test data are shown in Table 1 along with some calculated ADMET properties. Table 1 Activity data and ADMET parameter predictions.As a service to our customers we are providing this early version of the manuscript. such as toxoplasmic encephalitis, chorioretinitis, and death.8, 9 Infection acquired during pregnancy can be especially severe; it can transmit from mother to the fetus, leading to congenital toxoplasmosis which may result in abortion, neonatal death, or fetal abnormalities.2, 10C16 Currently, there is no vaccine available to prevent human infection from this pathogen. Antifolate agents, sulfadiazine and pyrimethamine, are two primary medicines for treatment of infection in humans.2, 15 Although these medicines are effective against tachyzoites in the acute stage of the disease, they do not eradicate encysted, latent bradyzoites. Furthermore, these therapies can be associated with side effects such as bone marrow depression, hypersensitivity and skin rashes.15, 16 There is an urgent need to develop new anti-medicines that are both efficacious and nontoxic to humans. One attractive target for chemotherapeutic intervention against apicomplexan parasites is the prokaryotic-like type II fatty acid biosynthesis (FAS-II) pathway.17C21 In and studies.21 Type II FAS is fundamentally divergent from the analogous FAS I pathway Rabbit polyclonal to ZNF317 in mammals. In eukaryotes, fatty acid biosynthetic enzymes integrate on a single multifunctional polypeptide (FASI), whereas fatty acid synthesis in prokaryotes utilizes a set of distinct enzymes composing the FAS-II pathway.22 Fatty acid biosynthesis is an iterative process beginning with condensation of acetyl-Coenzyme A (acetyl-CoA) with a growing fatty acid chain. In parasites with an IC50 value of ~200nM.18 Although triclosan is a potent inhibitor of TgENR, the diphenyl ether has low water solubility and a high ClogP value. Another major challenge for the development of medicines against targets which reside within the apicoplast of apicomplexan parasites is the need for the inhibitors to cross the four membranes of the parasite-specific organelle, in addition to the barriers set by both host cell and the parasite.27 In order to overcome these structural drawbacks and address the uptake problem, structure-based modification of triclosan was directed by improving the ADMET (absorption, distribution, metabolism, excretion and toxicity) profiles with special focus on the increase of aqueous solubility and permeability. We have previously shown that the A-ring of triclosan can be modified to exploit an additional space at the base of the inhibitor binding pocket.28 Furthermore, we have shown that substitution on the B-ring may also be tolerated to produce effective triclosan analogues {Stec et al., and using methods that have been described in the recent literature28, 33 For comparison of parasite burden between treatment groups, analysis of variance (ANOVA) was performed with group and run as factors. 3-Methyl-2-oxovaleric acid Due to evidence of non-normality, natural log-transformed parasite burden was used in the analysis.(Also, see Supplementary Material). The crystal structure of TgENR in complex with NAD+ and triclosan,34 showed that the 4-chloro phenoxy ring (A-ring) of triclosan participates in a – stacking interaction with NAD+, and a hydrogen bond forms between the hydroxyl group and Tyr189. However, the 2,4-dichlorophenoxy ring (B-ring) engages only in van der Waals interactions within a pocket encompassed by the peptide backbone of residues Leu128 to Ala131, the pyrophosphate and nicotinamide moieties of NAD+, and the side chains of Val134, Met193, Ala231 and Ile235. Moreover, there remains some additional space around the B-ring that could be exploited. In particular the B-ring is exposed to the outside solvent via a channel which would allow the fatty acyl substrate attached to the acyl carrier protein to enter the active site (Fig. 1A). Open in a separate window Figure 1 (A) The TgENR/NAD+/triclosan crystal structure shown in a cartoon representation covered by a transparent surface showing the channel which leads from the triclosan inhibitor to the outside solvent. TgENR Asn130 which has been targeted for the design of new inhibitors is shown in stick format close to the B-ring of triclosan. (B) A multiple sequence alignment in the vicinity of TgENR Asn130 (shown by an asterisk) of a number of parasitic and plant ENR enzymes showing full sequence conservation across parasite and plant families. In view of the space around the B-ring which could be exploited, we devised a modification strategy to change the B- ring, incorporating additional polar groups to optimize the physicochemical properties (such as permeability and solubility) while keeping the A-ring intact. These modifications were guided by computer-aided ADMET prediction (ADMET suite from ACD/labs). In total, 19 triclosan analogs have been designed with either substitutions at the.