Target-based discovery of novel inhibitors of enzymatic targets from Wolbachia endosymbionts and evaluation as antifilarial drug candidates
Target-based discovery of novel inhibitors of enzymatic targets from Wolbachia endosymbionts and evaluation as antifilarial drug candidates
Main document (4.8MB)Type of Scholarly Publication
DissertationDate of Exam
29.05.2013Date of Publication
24.07.2013Advisor
Hörauf, AchimCo-Referee
Famulok, MichaelMetadata
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Abstract
Filarial nematodes are the causative agents of vector-borne diseases known as lymphatic filariasis and onchocerciasis. More than 150 million inhabitants of endemic tropical and subtropical countries are infected and may develop severe and chronic pathologies such as lymphedema, hydrocele or river blindness. The lack of adulticidal activity and the development of resistance against current antifilarial drugs enforces the development of new antifilarial agents in order to finally cure and eradicate these diseases. Annotation of the genome of B. malayi, a causative agent of lymphatic filariasis, and its Wolbachia endosymbiont, have revealed a variety of essential endobacterial biochemical pathways that may be exploited as antifilarial drug targets.
On this background, my study aimed to discover novel inhibitors of two enzymatic targets of Wolbachia: One is the glycolytic/gluconeogenetic enzyme pyruvate phosphate dikinase (PPDK) that catalyzes the reversible conversion of phosphoenol pyruvate to pyruvate. The other is δ-aminolevulinic acid dehydratase (ALAD) which converts 5-aminolevulinic acid to porphobilinogen as the first common step of heme biosynthesis. For ALAD, a Zn2+-dependent human ortholog exists (hALAD) that is phylogenetically different from the Mg2+-responsive endobacterial enzyme (wALAD).
High-throughput compatible screening assays were established for both recombinantly expressed proteins and screened against a diversity-based compound library comprising ~18,000 drug-like small molecules for the identification of novel inhibitors and potential drug lead candidates. For wALAD, a cluster of novel tri-substituted benzimidazole-5-carboxylic acid structures was discovered that specifically bound to and inhibited the Mg2+-responsive wALAD with little effect on the human enzyme. For the most potent compound, termed wALADin1 (IC50 = 11 μM), a mixed competitive/non-competitive inhibitory mechanism was determined that also involves antagonism with the (apparently allosteric) activation of wALAD by Mg2+. While the plant/chloroplast ALAD orhtholog of pea is also potently inhibited by wALADin1, this compound stimulates enzymatic activity of several Mg2+ -responsive γ-proteobacterial orthologs suggesting that wALADins are, in general, (putatively allosteric) modulators of ALAD activity.
Proof-of-principle antifilarial activity of wALADin1 was demonstrated using the rodent filarial nematode L. sigmodontis in an ex vivo setup (IC50 < 125 µM) where wALADin1 induced a low-motility phenotype and a decline in worm viability. The specificity and Wolbachia-dependency of this effect was corroborated by control experiments with a Wolbachia-free rodent filarial. No antifilarial effect could be detected for wALADin1 in the course of L. sigmodontis infection in mice in vivo, presumably as a result of pharmakokinetic shortcomings.
Essential chemical features required for inhibitory potency and species-selectivity of wALADin-benzimidazoles were delineated in Structure-Activity-Relationship studies. Further inhibitory chemotypes were identified including a tricyclic quinoline derivative (wALADin2) with improved potency and specificity that acted by a related but not identical inhibitory mechanism. Finally, wALADin1 and, with even higher potency, two of its derivatives elicited antiprotozoal activity against blood stage Plasmodium falciparum (IC50 ~ 7 – 40 µM) but not against the related apicomplexan parasite Toxoplasma gondii.
On this background, my study aimed to discover novel inhibitors of two enzymatic targets of Wolbachia: One is the glycolytic/gluconeogenetic enzyme pyruvate phosphate dikinase (PPDK) that catalyzes the reversible conversion of phosphoenol pyruvate to pyruvate. The other is δ-aminolevulinic acid dehydratase (ALAD) which converts 5-aminolevulinic acid to porphobilinogen as the first common step of heme biosynthesis. For ALAD, a Zn2+-dependent human ortholog exists (hALAD) that is phylogenetically different from the Mg2+-responsive endobacterial enzyme (wALAD).
High-throughput compatible screening assays were established for both recombinantly expressed proteins and screened against a diversity-based compound library comprising ~18,000 drug-like small molecules for the identification of novel inhibitors and potential drug lead candidates. For wALAD, a cluster of novel tri-substituted benzimidazole-5-carboxylic acid structures was discovered that specifically bound to and inhibited the Mg2+-responsive wALAD with little effect on the human enzyme. For the most potent compound, termed wALADin1 (IC50 = 11 μM), a mixed competitive/non-competitive inhibitory mechanism was determined that also involves antagonism with the (apparently allosteric) activation of wALAD by Mg2+. While the plant/chloroplast ALAD orhtholog of pea is also potently inhibited by wALADin1, this compound stimulates enzymatic activity of several Mg2+ -responsive γ-proteobacterial orthologs suggesting that wALADins are, in general, (putatively allosteric) modulators of ALAD activity.
Proof-of-principle antifilarial activity of wALADin1 was demonstrated using the rodent filarial nematode L. sigmodontis in an ex vivo setup (IC50 < 125 µM) where wALADin1 induced a low-motility phenotype and a decline in worm viability. The specificity and Wolbachia-dependency of this effect was corroborated by control experiments with a Wolbachia-free rodent filarial. No antifilarial effect could be detected for wALADin1 in the course of L. sigmodontis infection in mice in vivo, presumably as a result of pharmakokinetic shortcomings.
Essential chemical features required for inhibitory potency and species-selectivity of wALADin-benzimidazoles were delineated in Structure-Activity-Relationship studies. Further inhibitory chemotypes were identified including a tricyclic quinoline derivative (wALADin2) with improved potency and specificity that acted by a related but not identical inhibitory mechanism. Finally, wALADin1 and, with even higher potency, two of its derivatives elicited antiprotozoal activity against blood stage Plasmodium falciparum (IC50 ~ 7 – 40 µM) but not against the related apicomplexan parasite Toxoplasma gondii.
Classification (DDC)
500 NaturwissenschaftenLentz, Christian Stephan: Target-based discovery of novel inhibitors of enzymatic targets from Wolbachia endosymbionts and evaluation as antifilarial drug candidates. - Bonn, 2013. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-32648
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-32648
@phdthesis{handle:20.500.11811/5710,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-32648,
author = {{Christian Stephan Lentz}},
title = {Target-based discovery of novel inhibitors of enzymatic targets from Wolbachia endosymbionts and evaluation as antifilarial drug candidates},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2013,
month = jul,
note = {Filarial nematodes are the causative agents of vector-borne diseases known as lymphatic filariasis and onchocerciasis. More than 150 million inhabitants of endemic tropical and subtropical countries are infected and may develop severe and chronic pathologies such as lymphedema, hydrocele or river blindness. The lack of adulticidal activity and the development of resistance against current antifilarial drugs enforces the development of new antifilarial agents in order to finally cure and eradicate these diseases. Annotation of the genome of B. malayi, a causative agent of lymphatic filariasis, and its Wolbachia endosymbiont, have revealed a variety of essential endobacterial biochemical pathways that may be exploited as antifilarial drug targets.
On this background, my study aimed to discover novel inhibitors of two enzymatic targets of Wolbachia: One is the glycolytic/gluconeogenetic enzyme pyruvate phosphate dikinase (PPDK) that catalyzes the reversible conversion of phosphoenol pyruvate to pyruvate. The other is δ-aminolevulinic acid dehydratase (ALAD) which converts 5-aminolevulinic acid to porphobilinogen as the first common step of heme biosynthesis. For ALAD, a Zn2+-dependent human ortholog exists (hALAD) that is phylogenetically different from the Mg2+-responsive endobacterial enzyme (wALAD).
High-throughput compatible screening assays were established for both recombinantly expressed proteins and screened against a diversity-based compound library comprising ~18,000 drug-like small molecules for the identification of novel inhibitors and potential drug lead candidates. For wALAD, a cluster of novel tri-substituted benzimidazole-5-carboxylic acid structures was discovered that specifically bound to and inhibited the Mg2+-responsive wALAD with little effect on the human enzyme. For the most potent compound, termed wALADin1 (IC50 = 11 μM), a mixed competitive/non-competitive inhibitory mechanism was determined that also involves antagonism with the (apparently allosteric) activation of wALAD by Mg2+. While the plant/chloroplast ALAD orhtholog of pea is also potently inhibited by wALADin1, this compound stimulates enzymatic activity of several Mg2+ -responsive γ-proteobacterial orthologs suggesting that wALADins are, in general, (putatively allosteric) modulators of ALAD activity.
Proof-of-principle antifilarial activity of wALADin1 was demonstrated using the rodent filarial nematode L. sigmodontis in an ex vivo setup (IC50 < 125 µM) where wALADin1 induced a low-motility phenotype and a decline in worm viability. The specificity and Wolbachia-dependency of this effect was corroborated by control experiments with a Wolbachia-free rodent filarial. No antifilarial effect could be detected for wALADin1 in the course of L. sigmodontis infection in mice in vivo, presumably as a result of pharmakokinetic shortcomings.
Essential chemical features required for inhibitory potency and species-selectivity of wALADin-benzimidazoles were delineated in Structure-Activity-Relationship studies. Further inhibitory chemotypes were identified including a tricyclic quinoline derivative (wALADin2) with improved potency and specificity that acted by a related but not identical inhibitory mechanism. Finally, wALADin1 and, with even higher potency, two of its derivatives elicited antiprotozoal activity against blood stage Plasmodium falciparum (IC50 ~ 7 – 40 µM) but not against the related apicomplexan parasite Toxoplasma gondii.},
url = {https://hdl.handle.net/20.500.11811/5710}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-32648,
author = {{Christian Stephan Lentz}},
title = {Target-based discovery of novel inhibitors of enzymatic targets from Wolbachia endosymbionts and evaluation as antifilarial drug candidates},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2013,
month = jul,
note = {Filarial nematodes are the causative agents of vector-borne diseases known as lymphatic filariasis and onchocerciasis. More than 150 million inhabitants of endemic tropical and subtropical countries are infected and may develop severe and chronic pathologies such as lymphedema, hydrocele or river blindness. The lack of adulticidal activity and the development of resistance against current antifilarial drugs enforces the development of new antifilarial agents in order to finally cure and eradicate these diseases. Annotation of the genome of B. malayi, a causative agent of lymphatic filariasis, and its Wolbachia endosymbiont, have revealed a variety of essential endobacterial biochemical pathways that may be exploited as antifilarial drug targets.
On this background, my study aimed to discover novel inhibitors of two enzymatic targets of Wolbachia: One is the glycolytic/gluconeogenetic enzyme pyruvate phosphate dikinase (PPDK) that catalyzes the reversible conversion of phosphoenol pyruvate to pyruvate. The other is δ-aminolevulinic acid dehydratase (ALAD) which converts 5-aminolevulinic acid to porphobilinogen as the first common step of heme biosynthesis. For ALAD, a Zn2+-dependent human ortholog exists (hALAD) that is phylogenetically different from the Mg2+-responsive endobacterial enzyme (wALAD).
High-throughput compatible screening assays were established for both recombinantly expressed proteins and screened against a diversity-based compound library comprising ~18,000 drug-like small molecules for the identification of novel inhibitors and potential drug lead candidates. For wALAD, a cluster of novel tri-substituted benzimidazole-5-carboxylic acid structures was discovered that specifically bound to and inhibited the Mg2+-responsive wALAD with little effect on the human enzyme. For the most potent compound, termed wALADin1 (IC50 = 11 μM), a mixed competitive/non-competitive inhibitory mechanism was determined that also involves antagonism with the (apparently allosteric) activation of wALAD by Mg2+. While the plant/chloroplast ALAD orhtholog of pea is also potently inhibited by wALADin1, this compound stimulates enzymatic activity of several Mg2+ -responsive γ-proteobacterial orthologs suggesting that wALADins are, in general, (putatively allosteric) modulators of ALAD activity.
Proof-of-principle antifilarial activity of wALADin1 was demonstrated using the rodent filarial nematode L. sigmodontis in an ex vivo setup (IC50 < 125 µM) where wALADin1 induced a low-motility phenotype and a decline in worm viability. The specificity and Wolbachia-dependency of this effect was corroborated by control experiments with a Wolbachia-free rodent filarial. No antifilarial effect could be detected for wALADin1 in the course of L. sigmodontis infection in mice in vivo, presumably as a result of pharmakokinetic shortcomings.
Essential chemical features required for inhibitory potency and species-selectivity of wALADin-benzimidazoles were delineated in Structure-Activity-Relationship studies. Further inhibitory chemotypes were identified including a tricyclic quinoline derivative (wALADin2) with improved potency and specificity that acted by a related but not identical inhibitory mechanism. Finally, wALADin1 and, with even higher potency, two of its derivatives elicited antiprotozoal activity against blood stage Plasmodium falciparum (IC50 ~ 7 – 40 µM) but not against the related apicomplexan parasite Toxoplasma gondii.},
url = {https://hdl.handle.net/20.500.11811/5710}
}
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