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Dinesh V Patel - One of the best experts on this subject based on the ideXlab platform.
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peptide deformylase inhibitors as antibacterial agents identification of vrc3375 a proline 3 alkylsuccinyl hydroxamate derivative by using an integrated combinatorial and medicinal chemistry approach
Antimicrobial Agents and Chemotherapy, 2004Co-Authors: Dawn Chen, C J Hackbarth, Zhijie Ni, Charlotte Wu, Wen Wang, Rakesh K Jain, Y He, Kathryn Rene Bracken, Beat Weidmann, Dinesh V PatelAbstract:Although the study of microbial genomes has revealed an abundance of potentially useful targets, so far little has resulted from this much-heralded effort. Instead, most newly disclosed antibacterial agents target known enzymes that were identified through the use of classical biological methods. Witness the selection of posters recently presented at the “New Antimicrobial Agents and New Research Technology” section of the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy: 18 proteins were discussed as targets for antibacterial drug discovery; all of these were discovered through standard microbiology and biochemistry methods. One such target that has received much attention lately is the bacterial peptide deformylase (PDF) (EC 3.5.1.31). The protein synthesis processes for bacterial and mammalian cells are very similar. Both utilize the same amino acids and codons and share the same mechanism for elongation. However, a major difference between bacterial protein synthesis and mammalian cytosol protein synthesis is the use of formylmethionine as the initiator (19, 20). Unlike cytosol protein synthesis in mammalian cells, which is initiated with methionine, protein synthesis in bacteria is initiated with N-Formylmethionine (2, 16), which is generated through enzymatic transformylation of methionyl-tRNA by formylmethionine tRNA transferase. The N-formyl methionine of the nascent protein in bacteria is removed by the sequential action of PDF and a methionine amino peptidase in order to afford the mature protein (2, 21). This formylation-deformylation cycle is essential for bacterial growth and is conserved among all studied bacterial species. Previous reports indicate that this cycle is not required for mammalian cells (for reviews, see references 7 and 31). The specific bacterial requirement for PDF in protein synthesis provides a rational basis for selectivity, making it an attractive drug discovery target. Although the in vitro development of bypass-resistant mutants in some bacterial organisms casts a shadow on the potential of using PDF inhibitors as antibacterial drugs, in vivo studies suggest that such mutants are much less virulent (for a detailed review, refer to Yuan et al. [31]). PDF activity was first reported by Adams in 1968 (1). This enzyme was not fully characterized until the early 1990s, when the Escherichia coli deformylase gene, def, was cloned (23) and when PDF was subsequently overproduced in E. coli (22, 23). Bacterial PDF utilizes a Fe2+ ion as the catalytic metal ion (8, 25, 26), but the ferrous ion in PDF is very unstable and can be quickly and irreversibly oxidized to the ferric species, resulting in an inactive enzyme (27). However, the ferrous ion can be replaced with a divalent nickel ion in vitro, resulting in much greater enzyme stability with little loss of enzyme activity (8). Since PDF is a metalloprotease, one possible approach to designing inhibitors consists of having a nonspecific chelating pharmacophore that binds to the catalytic metal ion and is coupled with a second moiety that binds to the active site, thus correctly positioning the chelator and providing the necessary selectivity and physicochemical properties. Such an approach has been successfully applied to the design of inhibitors of many other therapeutically important metalloproteases, the prime example being the angiotensin converting enzyme, followed more recently by the matrix metalloproteases (15). Recently, actinonin, a naturally occurring antibiotic with a hydroxamate moiety and a tripeptide binding domain, was shown to be a potent PDF inhibitor (5). Several three-dimensional studies of PDF-actinonin complexes suggest that the hydroxamate moiety of the inhibitor binds to and chelates the active center metal ion, while the tripeptide domain fits into the S1′-S3′ pocket of the enzyme (9). Using mechanistic information about the reaction catalyzed by PDF, together with an understanding of the general principles of metalloprotease inhibition, others have constructed several chelator-based inhibitor libraries according to the generic PDF inhibitor structure (Fig. (Fig.1)1) (31). In these libraries, X represents a chelating pharmacophore element that can bind to the metal ion at the active center of PDF, the N-butyl group mimics the methionine side chain, and P2′ and P3′ are domains of the inhibitor that can provide additional binding energy, selectivity, and favorable pharmacokinetic properties. These libraries were tested for PDF inhibition, antibacterial activity, and cytotoxicity. In this report, we describe the construction of such libraries, explore the inferred structure-activity relationships (SARs) of the resulting compounds, and report the subsequent identification of VRC3375, a unique PDF inhibitor-based broad-spectrum antibacterial agent with oral efficacy. FIG. 1. A generic PDF inhibitor structure. In the structure, X corresponds to a pharmacophore element capable of chelating metal ions. X attaches to a 2-substituted hexanoyl, which mimics the transition state of the hydrolysis of formyl-methionine.
Dawn Chen - One of the best experts on this subject based on the ideXlab platform.
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peptide deformylase inhibitors as antibacterial agents identification of vrc3375 a proline 3 alkylsuccinyl hydroxamate derivative by using an integrated combinatorial and medicinal chemistry approach
Antimicrobial Agents and Chemotherapy, 2004Co-Authors: Dawn Chen, C J Hackbarth, Zhijie Ni, Charlotte Wu, Wen Wang, Rakesh K Jain, Y He, Kathryn Rene Bracken, Beat Weidmann, Dinesh V PatelAbstract:Although the study of microbial genomes has revealed an abundance of potentially useful targets, so far little has resulted from this much-heralded effort. Instead, most newly disclosed antibacterial agents target known enzymes that were identified through the use of classical biological methods. Witness the selection of posters recently presented at the “New Antimicrobial Agents and New Research Technology” section of the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy: 18 proteins were discussed as targets for antibacterial drug discovery; all of these were discovered through standard microbiology and biochemistry methods. One such target that has received much attention lately is the bacterial peptide deformylase (PDF) (EC 3.5.1.31). The protein synthesis processes for bacterial and mammalian cells are very similar. Both utilize the same amino acids and codons and share the same mechanism for elongation. However, a major difference between bacterial protein synthesis and mammalian cytosol protein synthesis is the use of formylmethionine as the initiator (19, 20). Unlike cytosol protein synthesis in mammalian cells, which is initiated with methionine, protein synthesis in bacteria is initiated with N-Formylmethionine (2, 16), which is generated through enzymatic transformylation of methionyl-tRNA by formylmethionine tRNA transferase. The N-formyl methionine of the nascent protein in bacteria is removed by the sequential action of PDF and a methionine amino peptidase in order to afford the mature protein (2, 21). This formylation-deformylation cycle is essential for bacterial growth and is conserved among all studied bacterial species. Previous reports indicate that this cycle is not required for mammalian cells (for reviews, see references 7 and 31). The specific bacterial requirement for PDF in protein synthesis provides a rational basis for selectivity, making it an attractive drug discovery target. Although the in vitro development of bypass-resistant mutants in some bacterial organisms casts a shadow on the potential of using PDF inhibitors as antibacterial drugs, in vivo studies suggest that such mutants are much less virulent (for a detailed review, refer to Yuan et al. [31]). PDF activity was first reported by Adams in 1968 (1). This enzyme was not fully characterized until the early 1990s, when the Escherichia coli deformylase gene, def, was cloned (23) and when PDF was subsequently overproduced in E. coli (22, 23). Bacterial PDF utilizes a Fe2+ ion as the catalytic metal ion (8, 25, 26), but the ferrous ion in PDF is very unstable and can be quickly and irreversibly oxidized to the ferric species, resulting in an inactive enzyme (27). However, the ferrous ion can be replaced with a divalent nickel ion in vitro, resulting in much greater enzyme stability with little loss of enzyme activity (8). Since PDF is a metalloprotease, one possible approach to designing inhibitors consists of having a nonspecific chelating pharmacophore that binds to the catalytic metal ion and is coupled with a second moiety that binds to the active site, thus correctly positioning the chelator and providing the necessary selectivity and physicochemical properties. Such an approach has been successfully applied to the design of inhibitors of many other therapeutically important metalloproteases, the prime example being the angiotensin converting enzyme, followed more recently by the matrix metalloproteases (15). Recently, actinonin, a naturally occurring antibiotic with a hydroxamate moiety and a tripeptide binding domain, was shown to be a potent PDF inhibitor (5). Several three-dimensional studies of PDF-actinonin complexes suggest that the hydroxamate moiety of the inhibitor binds to and chelates the active center metal ion, while the tripeptide domain fits into the S1′-S3′ pocket of the enzyme (9). Using mechanistic information about the reaction catalyzed by PDF, together with an understanding of the general principles of metalloprotease inhibition, others have constructed several chelator-based inhibitor libraries according to the generic PDF inhibitor structure (Fig. (Fig.1)1) (31). In these libraries, X represents a chelating pharmacophore element that can bind to the metal ion at the active center of PDF, the N-butyl group mimics the methionine side chain, and P2′ and P3′ are domains of the inhibitor that can provide additional binding energy, selectivity, and favorable pharmacokinetic properties. These libraries were tested for PDF inhibition, antibacterial activity, and cytotoxicity. In this report, we describe the construction of such libraries, explore the inferred structure-activity relationships (SARs) of the resulting compounds, and report the subsequent identification of VRC3375, a unique PDF inhibitor-based broad-spectrum antibacterial agent with oral efficacy. FIG. 1. A generic PDF inhibitor structure. In the structure, X corresponds to a pharmacophore element capable of chelating metal ions. X attaches to a 2-substituted hexanoyl, which mimics the transition state of the hydrolysis of formyl-methionine.
P Soucek - One of the best experts on this subject based on the ideXlab platform.
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Expression of cytochrome P450 2A6 in Escherichia coli: purification, spectral and catalytic characterization, and preparation of polyclonal antibodies.
Archives of biochemistry and biophysics, 1999Co-Authors: P SoucekAbstract:Cytochrome P450 (CYP) 2A6 is the principal human enzyme catalyzing coumarin 7-hydroxylation and is known to be involved in the metabolism of halothane, nicotine, and metabolic activation of butadiene and nitrosamines. In this paper expression of CYP2A6 in Escherichia coli is reported. In order to achieve expression, the N-terminus of protein was modified by PCR mutagenesis. The N-terminal variant with only a single amino acid change showed expression of 210 nmol of CYP2A6/liter of culture. Recombinant CYP2A6 protein was purified to electrophoretic homogeneity and further characterized. Absolute spectra were typical for CYP proteins and indicated low spin characteristics of isolated protein. Due to a hydrophobic segment the N-terminal amino acid sequence of recombinant CYP2A6 was blocked. The N-terminal formylmethionine block was removed by mild acid treatment. Purified CYP2A6 had good catalytic activity toward marker substrate coumarin in a reconstituted system (K(m) = 1.48 +/- 0.37 microM, V(max) = 3.36 +/- 0.18 nmol product/min/nmol CYP). Its activity in the reconstituted system was stimulated by the presence of cytochrome b(5) and glutathione. CYP2A6 was shown to metabolize chlorzoxazone in the reconstituted system with activity of 0.32 nmol of product/min/nmol of CYP, and thus caution should be taken when interpretation of CYP2E1 in vivo phenotyping data is performed. Rabbit polyclonal antibodies were produced against recombinant CYP2A6 and proved to be very useful for immunoblotting and immunoinhibition studies. Availability of this expression system and specific antibodies should facilitate characterization of the role of CYP2A6 in the metabolism of chemicals and in the study biological relevance of genetic polymorphisms of this enzyme.
C J Hackbarth - One of the best experts on this subject based on the ideXlab platform.
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peptide deformylase inhibitors as antibacterial agents identification of vrc3375 a proline 3 alkylsuccinyl hydroxamate derivative by using an integrated combinatorial and medicinal chemistry approach
Antimicrobial Agents and Chemotherapy, 2004Co-Authors: Dawn Chen, C J Hackbarth, Zhijie Ni, Charlotte Wu, Wen Wang, Rakesh K Jain, Y He, Kathryn Rene Bracken, Beat Weidmann, Dinesh V PatelAbstract:Although the study of microbial genomes has revealed an abundance of potentially useful targets, so far little has resulted from this much-heralded effort. Instead, most newly disclosed antibacterial agents target known enzymes that were identified through the use of classical biological methods. Witness the selection of posters recently presented at the “New Antimicrobial Agents and New Research Technology” section of the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy: 18 proteins were discussed as targets for antibacterial drug discovery; all of these were discovered through standard microbiology and biochemistry methods. One such target that has received much attention lately is the bacterial peptide deformylase (PDF) (EC 3.5.1.31). The protein synthesis processes for bacterial and mammalian cells are very similar. Both utilize the same amino acids and codons and share the same mechanism for elongation. However, a major difference between bacterial protein synthesis and mammalian cytosol protein synthesis is the use of formylmethionine as the initiator (19, 20). Unlike cytosol protein synthesis in mammalian cells, which is initiated with methionine, protein synthesis in bacteria is initiated with N-Formylmethionine (2, 16), which is generated through enzymatic transformylation of methionyl-tRNA by formylmethionine tRNA transferase. The N-formyl methionine of the nascent protein in bacteria is removed by the sequential action of PDF and a methionine amino peptidase in order to afford the mature protein (2, 21). This formylation-deformylation cycle is essential for bacterial growth and is conserved among all studied bacterial species. Previous reports indicate that this cycle is not required for mammalian cells (for reviews, see references 7 and 31). The specific bacterial requirement for PDF in protein synthesis provides a rational basis for selectivity, making it an attractive drug discovery target. Although the in vitro development of bypass-resistant mutants in some bacterial organisms casts a shadow on the potential of using PDF inhibitors as antibacterial drugs, in vivo studies suggest that such mutants are much less virulent (for a detailed review, refer to Yuan et al. [31]). PDF activity was first reported by Adams in 1968 (1). This enzyme was not fully characterized until the early 1990s, when the Escherichia coli deformylase gene, def, was cloned (23) and when PDF was subsequently overproduced in E. coli (22, 23). Bacterial PDF utilizes a Fe2+ ion as the catalytic metal ion (8, 25, 26), but the ferrous ion in PDF is very unstable and can be quickly and irreversibly oxidized to the ferric species, resulting in an inactive enzyme (27). However, the ferrous ion can be replaced with a divalent nickel ion in vitro, resulting in much greater enzyme stability with little loss of enzyme activity (8). Since PDF is a metalloprotease, one possible approach to designing inhibitors consists of having a nonspecific chelating pharmacophore that binds to the catalytic metal ion and is coupled with a second moiety that binds to the active site, thus correctly positioning the chelator and providing the necessary selectivity and physicochemical properties. Such an approach has been successfully applied to the design of inhibitors of many other therapeutically important metalloproteases, the prime example being the angiotensin converting enzyme, followed more recently by the matrix metalloproteases (15). Recently, actinonin, a naturally occurring antibiotic with a hydroxamate moiety and a tripeptide binding domain, was shown to be a potent PDF inhibitor (5). Several three-dimensional studies of PDF-actinonin complexes suggest that the hydroxamate moiety of the inhibitor binds to and chelates the active center metal ion, while the tripeptide domain fits into the S1′-S3′ pocket of the enzyme (9). Using mechanistic information about the reaction catalyzed by PDF, together with an understanding of the general principles of metalloprotease inhibition, others have constructed several chelator-based inhibitor libraries according to the generic PDF inhibitor structure (Fig. (Fig.1)1) (31). In these libraries, X represents a chelating pharmacophore element that can bind to the metal ion at the active center of PDF, the N-butyl group mimics the methionine side chain, and P2′ and P3′ are domains of the inhibitor that can provide additional binding energy, selectivity, and favorable pharmacokinetic properties. These libraries were tested for PDF inhibition, antibacterial activity, and cytotoxicity. In this report, we describe the construction of such libraries, explore the inferred structure-activity relationships (SARs) of the resulting compounds, and report the subsequent identification of VRC3375, a unique PDF inhibitor-based broad-spectrum antibacterial agent with oral efficacy. FIG. 1. A generic PDF inhibitor structure. In the structure, X corresponds to a pharmacophore element capable of chelating metal ions. X attaches to a 2-substituted hexanoyl, which mimics the transition state of the hydrolysis of formyl-methionine.
Zhijie Ni - One of the best experts on this subject based on the ideXlab platform.
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peptide deformylase inhibitors as antibacterial agents identification of vrc3375 a proline 3 alkylsuccinyl hydroxamate derivative by using an integrated combinatorial and medicinal chemistry approach
Antimicrobial Agents and Chemotherapy, 2004Co-Authors: Dawn Chen, C J Hackbarth, Zhijie Ni, Charlotte Wu, Wen Wang, Rakesh K Jain, Y He, Kathryn Rene Bracken, Beat Weidmann, Dinesh V PatelAbstract:Although the study of microbial genomes has revealed an abundance of potentially useful targets, so far little has resulted from this much-heralded effort. Instead, most newly disclosed antibacterial agents target known enzymes that were identified through the use of classical biological methods. Witness the selection of posters recently presented at the “New Antimicrobial Agents and New Research Technology” section of the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy: 18 proteins were discussed as targets for antibacterial drug discovery; all of these were discovered through standard microbiology and biochemistry methods. One such target that has received much attention lately is the bacterial peptide deformylase (PDF) (EC 3.5.1.31). The protein synthesis processes for bacterial and mammalian cells are very similar. Both utilize the same amino acids and codons and share the same mechanism for elongation. However, a major difference between bacterial protein synthesis and mammalian cytosol protein synthesis is the use of formylmethionine as the initiator (19, 20). Unlike cytosol protein synthesis in mammalian cells, which is initiated with methionine, protein synthesis in bacteria is initiated with N-Formylmethionine (2, 16), which is generated through enzymatic transformylation of methionyl-tRNA by formylmethionine tRNA transferase. The N-formyl methionine of the nascent protein in bacteria is removed by the sequential action of PDF and a methionine amino peptidase in order to afford the mature protein (2, 21). This formylation-deformylation cycle is essential for bacterial growth and is conserved among all studied bacterial species. Previous reports indicate that this cycle is not required for mammalian cells (for reviews, see references 7 and 31). The specific bacterial requirement for PDF in protein synthesis provides a rational basis for selectivity, making it an attractive drug discovery target. Although the in vitro development of bypass-resistant mutants in some bacterial organisms casts a shadow on the potential of using PDF inhibitors as antibacterial drugs, in vivo studies suggest that such mutants are much less virulent (for a detailed review, refer to Yuan et al. [31]). PDF activity was first reported by Adams in 1968 (1). This enzyme was not fully characterized until the early 1990s, when the Escherichia coli deformylase gene, def, was cloned (23) and when PDF was subsequently overproduced in E. coli (22, 23). Bacterial PDF utilizes a Fe2+ ion as the catalytic metal ion (8, 25, 26), but the ferrous ion in PDF is very unstable and can be quickly and irreversibly oxidized to the ferric species, resulting in an inactive enzyme (27). However, the ferrous ion can be replaced with a divalent nickel ion in vitro, resulting in much greater enzyme stability with little loss of enzyme activity (8). Since PDF is a metalloprotease, one possible approach to designing inhibitors consists of having a nonspecific chelating pharmacophore that binds to the catalytic metal ion and is coupled with a second moiety that binds to the active site, thus correctly positioning the chelator and providing the necessary selectivity and physicochemical properties. Such an approach has been successfully applied to the design of inhibitors of many other therapeutically important metalloproteases, the prime example being the angiotensin converting enzyme, followed more recently by the matrix metalloproteases (15). Recently, actinonin, a naturally occurring antibiotic with a hydroxamate moiety and a tripeptide binding domain, was shown to be a potent PDF inhibitor (5). Several three-dimensional studies of PDF-actinonin complexes suggest that the hydroxamate moiety of the inhibitor binds to and chelates the active center metal ion, while the tripeptide domain fits into the S1′-S3′ pocket of the enzyme (9). Using mechanistic information about the reaction catalyzed by PDF, together with an understanding of the general principles of metalloprotease inhibition, others have constructed several chelator-based inhibitor libraries according to the generic PDF inhibitor structure (Fig. (Fig.1)1) (31). In these libraries, X represents a chelating pharmacophore element that can bind to the metal ion at the active center of PDF, the N-butyl group mimics the methionine side chain, and P2′ and P3′ are domains of the inhibitor that can provide additional binding energy, selectivity, and favorable pharmacokinetic properties. These libraries were tested for PDF inhibition, antibacterial activity, and cytotoxicity. In this report, we describe the construction of such libraries, explore the inferred structure-activity relationships (SARs) of the resulting compounds, and report the subsequent identification of VRC3375, a unique PDF inhibitor-based broad-spectrum antibacterial agent with oral efficacy. FIG. 1. A generic PDF inhibitor structure. In the structure, X corresponds to a pharmacophore element capable of chelating metal ions. X attaches to a 2-substituted hexanoyl, which mimics the transition state of the hydrolysis of formyl-methionine.
