The Experts below are selected from a list of 318 Experts worldwide ranked by ideXlab platform
Joseph A. Kovacs - One of the best experts on this subject based on the ideXlab platform.
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at al. Interaction of sulfonamide and sulfone compounds with Toxoplasma gondii Dihydropteroate Synthase
2016Co-Authors: Carmen J. Allegra, Joseph A. Kovacs, Donna Boarman, J Beaver, Bruce A. Chabner, Paul Morrison, Henry MasurtAbstract:Toxoplasma gondii is a common protozoan disease that often causes life-threatening disease, particularly among patients with the acquired immunodeficiency syndrome. This study demonstrates that the Dihydropteroate Synthase in T. gondii is kinetically distinct from the enzyme characterized from other sources and can be highly purified with a high yield using sequential dye-affinity chromatography. Conditions have been identified that allow for stabilization of the purified enzyme, and its physical characteristics have been elucidated. The mo-lecular weight of the native protein was 125,000 and the pro-tein appeared to contain both Dihydropteroate Synthase and 6-hydroxymethyl-dihydropterin pyrophosphokinase activities. The sulfonamide class ofcompounds vary in inhibitory potency by more than three orders of magnitude. Sulfathiazole, sulfa-methoxazole, and sulfamethazine, with 50 % inhibitory concen-trations (IC50's) of 1.7, 2.7, and 5.7 MM, respectively, represent the most potent of this class of inhibitors. Several sulfone analogues, including dapsone, were identified as highly potent inhibitors with ICN's < 1 uM. The results of these cell-free experiments were corroborated by investigating the metabolic inhibition produced by the various inhibitors in intact organ-isms. The qualitative and quantitative relations among the in-hibitors were preserved in both the cell-free and intact cell assay systems. These studies suggest that the sulfones may be important therapeutic agents for the treatment of toxoplas-mosis. (J. Clin. Invest. 1990. 85:371-379.) antimetabolites. enzymology- protozoan * sulfonamide
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Mutational Analysis of Pneumocystis jirovecii Dihydropteroate Synthase and Dihydrofolate Reductase Genes in HIV-Infected Patients in China
Journal of clinical microbiology, 2014Co-Authors: Xilong Deng, Joseph A. Kovacs, Li Zhuo, Yun Lan, Zhaoxia Dai, Wanshan Chen, Weiping Cai, Xiaoping TangAbstract:We investigated Pneumocystis jirovecii Dihydropteroate Synthase (DHPS) and dihydrofolate reductase (DHFR) genes for mutations in 25 Chinese HIV-infected patients with P. jirovecii pneumonia. We identified DHPS mutations in 3 (12%) patients and DHFR mutations in 1 (4%) patient. The prevalence of DHPS and DHFR mutations in China remains low, as it does in other developing countries.
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Genetic Analysis of Multiple Loci Suggests that Mutations in the Pneumocystis carinii f. sp.hominis Dihydropteroate Synthase Gene Arose Independently in Multiple Strains
Antimicrobial agents and chemotherapy, 2001Co-Authors: Joseph A. KovacsAbstract:To determine if mutations in the Dihydropteroate Synthase (DHPS) gene of Pneumocystis carinii f. sp. hominis arose in a single strain that was subsequently widely disseminated, we examined four genomic regions of 22 P. carinii clinical isolates selected based on the absence or presence of mutations in the DHPS gene. By single-strand conformation polymorphism and DNA sequencing, we found varying genotypes for each of the four regions in isolates with DHPS mutations, suggesting that these mutations occurred independently in multiple strains of P. carinii. This suggests that exposure to sulfa will select for these mutations in diverse strains.
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Genetic divergence of the dihydrofolate reductase and Dihydropteroate Synthase genes in Pneumocystis carinii from 7 different host species.
The Journal of infectious diseases, 2001Co-Authors: Hiromi Imamichi, Antti Sukura, Joseph A. KovacsAbstract:To investigate the phylogenetic and therapeutic implications of the genetic divergence in the dihydrofolate reductase (DHFR) and Dihydropteroate Synthase (DHPS) genes among different Pneumocystis carinii strains, these 2 genes in P. carinii obtained from 7 different host species were sequenced. Pairwise comparison of the DHPS sequences demonstrated 6%–24% and 6%–30% divergence in the nucleotide and deduced amino acid sequences, respectively. The DHFR gene was even more divergent, with differences of 15%–34% and 18%–42% in the nucleotide and deduced amino acid sequences, respectively. Phylogenetic analysis of DHFR and DHPS sequences revealed that all P. carinii strains were confined within a distinct group that was closely related to ascomycete fungi and that human-derived P. carinii was most closely related to monkey-derived P. carinii. Recognizing the substantial differences in the DHFR and DHPS genes among P. carinii from different host species has important implications for drug discovery and the development of new diagnostic methods. Pneumocystis carinii is an important opportunistic pathogen that infects humans and a wide range of other mammalian host species. Each host species has its own special form(s) of P. carinii. It is well documented that various P. carinii organisms isolated from different host species are distinct in terms of antigenic epitopes, isoenzymes, chromosomal karyotypes, and DNA sequences of genetic loci. Furthermore, cross-infection experiments have shown that P. carinii isolated from one host species cannot infect another host species. These data have contributed to the view that P. carinii is host-species specific. Dihydrofolate reductase (DHFR) and Dihydropteroate Synthase (DHPS) are the targets of trimethoprim and sulfa drugs, respectively, which are widely used for treatment and prophylaxis of P. carinii pneumonia (PCP) in humans. A large body of information about these drugs and their targets has been
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Rapid detection of mutations in the human-derived Pneumocystis carinii Dihydropteroate Synthase gene associated with sulfa resistance.
Antimicrobial agents and chemotherapy, 2001Co-Authors: Joseph A. KovacsAbstract:Recent studies have shown that point mutations in the Dihydropteroate Synthase (DHPS) gene of human-derived Pneumocystis carinii are related to exposure to sulfa drugs and possibly represent the emergence of sulfa resistance. We developed a simple single-strand conformation polymorphism (SSCP) method to permit rapid detection of these mutations. With plasmid constructs, SSCP was able to detect as little as 10% of a minority population. The SSCP assay was compared to direct sequencing for typing the DHPS gene by examining 37 clinical isolates with known DHPS sequences and 41 clinical isolates with unknown DHPS sequences. The typing results were consistent between these two methods for all isolates except 11 in which mutations were detected by SSCP but not by direct sequencing. Sequencing of individual clones after subcloning confirmed the presence of mutations in a minority population as determined by SSCP. SSCP is a very simple and sensitive method for rapid identification of P. camii DHPS mutations.
Richard E. Lee - One of the best experts on this subject based on the ideXlab platform.
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Pterin-sulfa conjugates as Dihydropteroate Synthase inhibitors and antibacterial agents.
Bioorganic & medicinal chemistry letters, 2016Co-Authors: Ying Zhao, Mi-kyung Yun, Stephen W. White, William R. Shadrick, Miranda J. Wallace, Elizabeth C. Griffith, Richard E. LeeAbstract:The sulfonamide class of antibiotics has been in continuous use for over 70years. They are thought to act by directly inhibiting Dihydropteroate Synthase (DHPS), and also acting as prodrugs that sequester pterin pools by forming dead end pterin-sulfonamide conjugates. In this study, eight pterin-sulfonamide conjugates were synthesized using a novel synthetic strategy and their biochemical and microbiological properties were investigated. The conjugates were shown to competitively inhibit DHPS, and inhibition was enhanced by the presence of pyrophosphate that is crucial to catalysis and is known to promote an ordering of the DHPS active site. The co-crystal structure of Yersinia pestis DHPS bound to one of the more potent conjugates revealed a mode of binding that is similar to that of the enzymatic product analog pteroic acid. The antimicrobial activities of the pterin-sulfonamide conjugates were measured against Escherichia coli in the presence and absence of folate precursors and dependent metabolites. These results show that the conjugates have appreciable antibacterial activity and act by an on target, anti-folate pathway mechanism rather than as simple dead end products.
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Identification and characterization of an allosteric inhibitory site on Dihydropteroate Synthase.
ACS chemical biology, 2014Co-Authors: Dalia I. Hammoudeh, Mi-kyung Yun, Richard E. Lee, Donald Bashford, Elizabeth C. Griffith, M. Date, Weixing Zhang, Vincent A. Boyd, Ariele Viacava Follis, Stephen W. WhiteAbstract:The declining effectiveness of current antibiotics due to the emergence of resistant bacterial strains dictates a pressing need for novel classes of antimicrobial therapies, preferably against molecular sites other than those in which resistance mutations have developed. Dihydropteroate Synthase (DHPS) catalyzes a crucial step in the bacterial pathway of folic acid synthesis, a pathway that is absent in higher vertebrates. As the target of the sulfonamide class of drugs that were highly effective until resistance mutations arose, DHPS is known to be a valuable bacterial Achilles heel that is being further exploited for antibiotic development. Here, we report the discovery of the first known allosteric inhibitor of DHPS. NMR and crystallographic studies reveal that it engages a previously unknown binding site at the dimer interface. Kinetic data show that this inhibitor does not prevent substrate binding but rather exerts its effect at a later step in the catalytic cycle. Molecular dynamics simulations and...
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Catalysis and sulfa drug resistance in Dihydropteroate Synthase.
Science (New York N.Y.), 2012Co-Authors: Mi-kyung Yun, Ying Zhao, Richard E. Lee, M.b. Waddell, Antonio M. Ferreira, Donald Bashford, Stephen W. WhiteAbstract:The sulfonamide antibiotics inhibit Dihydropteroate Synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an S(N)1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active-site residues and reveals how sulfonamide resistance arises.
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Development of a pterin-based fluorescent probe for screening Dihydropteroate Synthase.
Bioconjugate chemistry, 2011Co-Authors: Ying Zhao, Sourav Das, Mi-kyung Yun, Stephen W. White, Elizabeth C. Griffith, Dalia I. Hammoudeh, Wenwei Lin, Taosheng Chen, Richard E. LeeAbstract:Dihydropteroate Synthase (DHPS) is the classical target of the sulfonamide class of antimicrobial agents, whose use has been limited by widespread resistance and pharmacological side effects. We have initiated a structure-based drug design approach for the development of novel DHPS inhibitors that bind to the highly conserved and structured pterin subsite rather than to the adjacent p-aminobenzoic acid binding pocket that is targeted by the sulfonamide class of antibiotics. To facilitate these studies, a robust pterin site-specific fluorescence polarization (FP) assay has been developed and is discussed herein. These studies include the design, synthesis, and characterization of two fluorescent probes, and the development and validation of a rapid DHPS FP assay. This assay has excellent DMSO tolerance and is highly reproducible as evidenced by a high Z′ factor. This assay offers significant advantages over traditional radiometric or phosphate release assays against this target, and is suitable for site-sp...
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Synthesis of bi-substrate state mimics of Dihydropteroate Synthase as potential inhibitors and molecular probes.
Bioorganic & medicinal chemistry, 2010Co-Authors: Kristopher G. Virga, Sourav Das, Ying Zhao, Mi-kyung Yun, Stephen W. White, Richard E. LeeAbstract:The increasing emergence of resistant bacteria drives us to design and develop new antimicrobial agents. Pursuant to that goal, a new targeting approach of the Dihydropteroate Synthase enzyme, which serves as the site of action for the sulfonamide class of antimicrobial agents, is being explored. Using structural information, a new class of transition state mimics has been designed and synthesized that have the capacity to bind to the pterin, phosphate and para-amino binding sites. The design, synthesis and evaluation of these compounds as inhibitors of Bacillus anthracis Dihydropteroate Synthase is described herein. Outcomes from this work have identified the first trivalent inhibitors of Dihydropteroate Synthase whose activity displayed slow binding inhibition. The most active compounds in this series contained an oxidized pterin ring. The binding of these inhibitors was modeled into the Dihydropteroate Synthase active site and demonstrated a good correlation with the observed bioassay data, as well as provided important insight for the future design of higher affinity transition state mimics.
Stephen W. White - One of the best experts on this subject based on the ideXlab platform.
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Pterin-sulfa conjugates as Dihydropteroate Synthase inhibitors and antibacterial agents.
Bioorganic & medicinal chemistry letters, 2016Co-Authors: Ying Zhao, Mi-kyung Yun, Stephen W. White, William R. Shadrick, Miranda J. Wallace, Elizabeth C. Griffith, Richard E. LeeAbstract:The sulfonamide class of antibiotics has been in continuous use for over 70years. They are thought to act by directly inhibiting Dihydropteroate Synthase (DHPS), and also acting as prodrugs that sequester pterin pools by forming dead end pterin-sulfonamide conjugates. In this study, eight pterin-sulfonamide conjugates were synthesized using a novel synthetic strategy and their biochemical and microbiological properties were investigated. The conjugates were shown to competitively inhibit DHPS, and inhibition was enhanced by the presence of pyrophosphate that is crucial to catalysis and is known to promote an ordering of the DHPS active site. The co-crystal structure of Yersinia pestis DHPS bound to one of the more potent conjugates revealed a mode of binding that is similar to that of the enzymatic product analog pteroic acid. The antimicrobial activities of the pterin-sulfonamide conjugates were measured against Escherichia coli in the presence and absence of folate precursors and dependent metabolites. These results show that the conjugates have appreciable antibacterial activity and act by an on target, anti-folate pathway mechanism rather than as simple dead end products.
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Identification and characterization of an allosteric inhibitory site on Dihydropteroate Synthase.
ACS chemical biology, 2014Co-Authors: Dalia I. Hammoudeh, Mi-kyung Yun, Richard E. Lee, Donald Bashford, Elizabeth C. Griffith, M. Date, Weixing Zhang, Vincent A. Boyd, Ariele Viacava Follis, Stephen W. WhiteAbstract:The declining effectiveness of current antibiotics due to the emergence of resistant bacterial strains dictates a pressing need for novel classes of antimicrobial therapies, preferably against molecular sites other than those in which resistance mutations have developed. Dihydropteroate Synthase (DHPS) catalyzes a crucial step in the bacterial pathway of folic acid synthesis, a pathway that is absent in higher vertebrates. As the target of the sulfonamide class of drugs that were highly effective until resistance mutations arose, DHPS is known to be a valuable bacterial Achilles heel that is being further exploited for antibiotic development. Here, we report the discovery of the first known allosteric inhibitor of DHPS. NMR and crystallographic studies reveal that it engages a previously unknown binding site at the dimer interface. Kinetic data show that this inhibitor does not prevent substrate binding but rather exerts its effect at a later step in the catalytic cycle. Molecular dynamics simulations and...
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Catalysis and sulfa drug resistance in Dihydropteroate Synthase.
Science (New York N.Y.), 2012Co-Authors: Mi-kyung Yun, Ying Zhao, Richard E. Lee, M.b. Waddell, Antonio M. Ferreira, Donald Bashford, Stephen W. WhiteAbstract:The sulfonamide antibiotics inhibit Dihydropteroate Synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an S(N)1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active-site residues and reveals how sulfonamide resistance arises.
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Development of a pterin-based fluorescent probe for screening Dihydropteroate Synthase.
Bioconjugate chemistry, 2011Co-Authors: Ying Zhao, Sourav Das, Mi-kyung Yun, Stephen W. White, Elizabeth C. Griffith, Dalia I. Hammoudeh, Wenwei Lin, Taosheng Chen, Richard E. LeeAbstract:Dihydropteroate Synthase (DHPS) is the classical target of the sulfonamide class of antimicrobial agents, whose use has been limited by widespread resistance and pharmacological side effects. We have initiated a structure-based drug design approach for the development of novel DHPS inhibitors that bind to the highly conserved and structured pterin subsite rather than to the adjacent p-aminobenzoic acid binding pocket that is targeted by the sulfonamide class of antibiotics. To facilitate these studies, a robust pterin site-specific fluorescence polarization (FP) assay has been developed and is discussed herein. These studies include the design, synthesis, and characterization of two fluorescent probes, and the development and validation of a rapid DHPS FP assay. This assay has excellent DMSO tolerance and is highly reproducible as evidenced by a high Z′ factor. This assay offers significant advantages over traditional radiometric or phosphate release assays against this target, and is suitable for site-sp...
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Synthesis of bi-substrate state mimics of Dihydropteroate Synthase as potential inhibitors and molecular probes.
Bioorganic & medicinal chemistry, 2010Co-Authors: Kristopher G. Virga, Sourav Das, Ying Zhao, Mi-kyung Yun, Stephen W. White, Richard E. LeeAbstract:The increasing emergence of resistant bacteria drives us to design and develop new antimicrobial agents. Pursuant to that goal, a new targeting approach of the Dihydropteroate Synthase enzyme, which serves as the site of action for the sulfonamide class of antimicrobial agents, is being explored. Using structural information, a new class of transition state mimics has been designed and synthesized that have the capacity to bind to the pterin, phosphate and para-amino binding sites. The design, synthesis and evaluation of these compounds as inhibitors of Bacillus anthracis Dihydropteroate Synthase is described herein. Outcomes from this work have identified the first trivalent inhibitors of Dihydropteroate Synthase whose activity displayed slow binding inhibition. The most active compounds in this series contained an oxidized pterin ring. The binding of these inhibitors was modeled into the Dihydropteroate Synthase active site and demonstrated a good correlation with the observed bioassay data, as well as provided important insight for the future design of higher affinity transition state mimics.
Mi-kyung Yun - One of the best experts on this subject based on the ideXlab platform.
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Pterin-sulfa conjugates as Dihydropteroate Synthase inhibitors and antibacterial agents.
Bioorganic & medicinal chemistry letters, 2016Co-Authors: Ying Zhao, Mi-kyung Yun, Stephen W. White, William R. Shadrick, Miranda J. Wallace, Elizabeth C. Griffith, Richard E. LeeAbstract:The sulfonamide class of antibiotics has been in continuous use for over 70years. They are thought to act by directly inhibiting Dihydropteroate Synthase (DHPS), and also acting as prodrugs that sequester pterin pools by forming dead end pterin-sulfonamide conjugates. In this study, eight pterin-sulfonamide conjugates were synthesized using a novel synthetic strategy and their biochemical and microbiological properties were investigated. The conjugates were shown to competitively inhibit DHPS, and inhibition was enhanced by the presence of pyrophosphate that is crucial to catalysis and is known to promote an ordering of the DHPS active site. The co-crystal structure of Yersinia pestis DHPS bound to one of the more potent conjugates revealed a mode of binding that is similar to that of the enzymatic product analog pteroic acid. The antimicrobial activities of the pterin-sulfonamide conjugates were measured against Escherichia coli in the presence and absence of folate precursors and dependent metabolites. These results show that the conjugates have appreciable antibacterial activity and act by an on target, anti-folate pathway mechanism rather than as simple dead end products.
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Identification and characterization of an allosteric inhibitory site on Dihydropteroate Synthase.
ACS chemical biology, 2014Co-Authors: Dalia I. Hammoudeh, Mi-kyung Yun, Richard E. Lee, Donald Bashford, Elizabeth C. Griffith, M. Date, Weixing Zhang, Vincent A. Boyd, Ariele Viacava Follis, Stephen W. WhiteAbstract:The declining effectiveness of current antibiotics due to the emergence of resistant bacterial strains dictates a pressing need for novel classes of antimicrobial therapies, preferably against molecular sites other than those in which resistance mutations have developed. Dihydropteroate Synthase (DHPS) catalyzes a crucial step in the bacterial pathway of folic acid synthesis, a pathway that is absent in higher vertebrates. As the target of the sulfonamide class of drugs that were highly effective until resistance mutations arose, DHPS is known to be a valuable bacterial Achilles heel that is being further exploited for antibiotic development. Here, we report the discovery of the first known allosteric inhibitor of DHPS. NMR and crystallographic studies reveal that it engages a previously unknown binding site at the dimer interface. Kinetic data show that this inhibitor does not prevent substrate binding but rather exerts its effect at a later step in the catalytic cycle. Molecular dynamics simulations and...
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Catalysis and sulfa drug resistance in Dihydropteroate Synthase.
Science (New York N.Y.), 2012Co-Authors: Mi-kyung Yun, Ying Zhao, Richard E. Lee, M.b. Waddell, Antonio M. Ferreira, Donald Bashford, Stephen W. WhiteAbstract:The sulfonamide antibiotics inhibit Dihydropteroate Synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an S(N)1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active-site residues and reveals how sulfonamide resistance arises.
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Development of a pterin-based fluorescent probe for screening Dihydropteroate Synthase.
Bioconjugate chemistry, 2011Co-Authors: Ying Zhao, Sourav Das, Mi-kyung Yun, Stephen W. White, Elizabeth C. Griffith, Dalia I. Hammoudeh, Wenwei Lin, Taosheng Chen, Richard E. LeeAbstract:Dihydropteroate Synthase (DHPS) is the classical target of the sulfonamide class of antimicrobial agents, whose use has been limited by widespread resistance and pharmacological side effects. We have initiated a structure-based drug design approach for the development of novel DHPS inhibitors that bind to the highly conserved and structured pterin subsite rather than to the adjacent p-aminobenzoic acid binding pocket that is targeted by the sulfonamide class of antibiotics. To facilitate these studies, a robust pterin site-specific fluorescence polarization (FP) assay has been developed and is discussed herein. These studies include the design, synthesis, and characterization of two fluorescent probes, and the development and validation of a rapid DHPS FP assay. This assay has excellent DMSO tolerance and is highly reproducible as evidenced by a high Z′ factor. This assay offers significant advantages over traditional radiometric or phosphate release assays against this target, and is suitable for site-sp...
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Synthesis of bi-substrate state mimics of Dihydropteroate Synthase as potential inhibitors and molecular probes.
Bioorganic & medicinal chemistry, 2010Co-Authors: Kristopher G. Virga, Sourav Das, Ying Zhao, Mi-kyung Yun, Stephen W. White, Richard E. LeeAbstract:The increasing emergence of resistant bacteria drives us to design and develop new antimicrobial agents. Pursuant to that goal, a new targeting approach of the Dihydropteroate Synthase enzyme, which serves as the site of action for the sulfonamide class of antimicrobial agents, is being explored. Using structural information, a new class of transition state mimics has been designed and synthesized that have the capacity to bind to the pterin, phosphate and para-amino binding sites. The design, synthesis and evaluation of these compounds as inhibitors of Bacillus anthracis Dihydropteroate Synthase is described herein. Outcomes from this work have identified the first trivalent inhibitors of Dihydropteroate Synthase whose activity displayed slow binding inhibition. The most active compounds in this series contained an oxidized pterin ring. The binding of these inhibitors was modeled into the Dihydropteroate Synthase active site and demonstrated a good correlation with the observed bioassay data, as well as provided important insight for the future design of higher affinity transition state mimics.
Ying Zhao - One of the best experts on this subject based on the ideXlab platform.
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Pterin-sulfa conjugates as Dihydropteroate Synthase inhibitors and antibacterial agents.
Bioorganic & medicinal chemistry letters, 2016Co-Authors: Ying Zhao, Mi-kyung Yun, Stephen W. White, William R. Shadrick, Miranda J. Wallace, Elizabeth C. Griffith, Richard E. LeeAbstract:The sulfonamide class of antibiotics has been in continuous use for over 70years. They are thought to act by directly inhibiting Dihydropteroate Synthase (DHPS), and also acting as prodrugs that sequester pterin pools by forming dead end pterin-sulfonamide conjugates. In this study, eight pterin-sulfonamide conjugates were synthesized using a novel synthetic strategy and their biochemical and microbiological properties were investigated. The conjugates were shown to competitively inhibit DHPS, and inhibition was enhanced by the presence of pyrophosphate that is crucial to catalysis and is known to promote an ordering of the DHPS active site. The co-crystal structure of Yersinia pestis DHPS bound to one of the more potent conjugates revealed a mode of binding that is similar to that of the enzymatic product analog pteroic acid. The antimicrobial activities of the pterin-sulfonamide conjugates were measured against Escherichia coli in the presence and absence of folate precursors and dependent metabolites. These results show that the conjugates have appreciable antibacterial activity and act by an on target, anti-folate pathway mechanism rather than as simple dead end products.
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Catalysis and sulfa drug resistance in Dihydropteroate Synthase.
Science (New York N.Y.), 2012Co-Authors: Mi-kyung Yun, Ying Zhao, Richard E. Lee, M.b. Waddell, Antonio M. Ferreira, Donald Bashford, Stephen W. WhiteAbstract:The sulfonamide antibiotics inhibit Dihydropteroate Synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an S(N)1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active-site residues and reveals how sulfonamide resistance arises.
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Development of a pterin-based fluorescent probe for screening Dihydropteroate Synthase.
Bioconjugate chemistry, 2011Co-Authors: Ying Zhao, Sourav Das, Mi-kyung Yun, Stephen W. White, Elizabeth C. Griffith, Dalia I. Hammoudeh, Wenwei Lin, Taosheng Chen, Richard E. LeeAbstract:Dihydropteroate Synthase (DHPS) is the classical target of the sulfonamide class of antimicrobial agents, whose use has been limited by widespread resistance and pharmacological side effects. We have initiated a structure-based drug design approach for the development of novel DHPS inhibitors that bind to the highly conserved and structured pterin subsite rather than to the adjacent p-aminobenzoic acid binding pocket that is targeted by the sulfonamide class of antibiotics. To facilitate these studies, a robust pterin site-specific fluorescence polarization (FP) assay has been developed and is discussed herein. These studies include the design, synthesis, and characterization of two fluorescent probes, and the development and validation of a rapid DHPS FP assay. This assay has excellent DMSO tolerance and is highly reproducible as evidenced by a high Z′ factor. This assay offers significant advantages over traditional radiometric or phosphate release assays against this target, and is suitable for site-sp...
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Synthesis of bi-substrate state mimics of Dihydropteroate Synthase as potential inhibitors and molecular probes.
Bioorganic & medicinal chemistry, 2010Co-Authors: Kristopher G. Virga, Sourav Das, Ying Zhao, Mi-kyung Yun, Stephen W. White, Richard E. LeeAbstract:The increasing emergence of resistant bacteria drives us to design and develop new antimicrobial agents. Pursuant to that goal, a new targeting approach of the Dihydropteroate Synthase enzyme, which serves as the site of action for the sulfonamide class of antimicrobial agents, is being explored. Using structural information, a new class of transition state mimics has been designed and synthesized that have the capacity to bind to the pterin, phosphate and para-amino binding sites. The design, synthesis and evaluation of these compounds as inhibitors of Bacillus anthracis Dihydropteroate Synthase is described herein. Outcomes from this work have identified the first trivalent inhibitors of Dihydropteroate Synthase whose activity displayed slow binding inhibition. The most active compounds in this series contained an oxidized pterin ring. The binding of these inhibitors was modeled into the Dihydropteroate Synthase active site and demonstrated a good correlation with the observed bioassay data, as well as provided important insight for the future design of higher affinity transition state mimics.