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Chengshu Wang - One of the best experts on this subject based on the ideXlab platform.
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Cyclosporine biosynthesis in Tolypocladium inflatum benefits fungal adaptation to the environment
mBio, 2018Co-Authors: Xiuqing Yang, Peng Feng, Ying Yin, Kathryn Bushley, Joseph W. Spatafora, Chengshu WangAbstract:The cycloundecapeptide cyclosporin A (CsA) was first isolated from the insect-pathogenic fungus Tolypocladium inflatum for its antifungal activity and later developed as an immunosuppressant drug. However, the full biosynthetic mechanism of CsA remains unknown and has puzzled researchers for decades. In this study, the biosynthetic gene cluster is suggested to include 12 genes encoding enzymes, including the nonribosomal peptide synthetase (NRPS) (SimA) responsible for assembling the 11 amino acid substrates of cyclosporine and a polyketide synthase (PKS) (SimG) to mediate the production of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-l-threonine (Bmt). Individual deletion of 10 genes, isolation of intermediates, and substrate feeding experiments show that Bmt is biosynthesized by three enzymes, including SimG, SimI, and SimJ. The substrate d-alanine is catalyzed from l-alanine by alanine racemase SimB. Gene cluster transcription is regulated by a putative basic leucine zipper (bZIP)-type protein encoded by the cluster gene SimL We also found that the cluster cyclophilin (SimC) and transporter (SimD) genes contribute to the tolerance of CsA in the CsA-producing fungus. We also found that cyclosporine production could enable the fungus to outcompete other fungi during cocultivation tests. Deletion of the CsA biosynthetic genes also impaired fungal virulence against insect hosts. Taking all the data together, in addition to proposing a biosynthetic pathway of cyclosporines, the results of this study suggest that CsA produced by this fungus might play important ecological roles in fungal environment interactions.IMPORTANCE The cyclopeptide cyclosporin A was first isolated from the filamentous fungus Tolypocladium inflatum showing antifungal activity and was later developed as an immunosuppressant drug. We report the biosynthetic mechanism of cyclosporines that are mediated by a cluster of genes encoding NRPS and PKS controlled by a bZIP-type transcriptional regulator. The two unusual amino acids Bmt and d-Ala are produced by the PKS pathway and alanine racemase, respectively. The cyclophilin and transporter genes jointly contribute to fungal self-protection against cyclosporines. Cyclosporine confers on T. inflatum the abilities to outcompete other fungi in competitive interactions and to facilitate fungal infection of insect hosts, which therefore benefits fungal adaptations to different environments.
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Cyclosporine Biosynthesis in Tolypocladium inflatum Benefits Fungal Adaptation to the Environment
American Society for Microbiology, 2018Co-Authors: Xiuqing Yang, Peng Feng, Ying Yin, Kathryn Bushley, Joseph W. Spatafora, Chengshu WangAbstract:The cyclopeptide cyclosporin A was first isolated from the filamentous fungus Tolypocladium inflatum showing antifungal activity and was later developed as an immunosuppressant drug. We report the biosynthetic mechanism of cyclosporines that are mediated by a cluster of genes encoding NRPS and PKS controlled by a bZIP-type transcriptional regulator. The two unusual amino acids Bmt and d-Ala are produced by the PKS pathway and alanine racemase, respectively. The cyclophilin and transporter genes jointly contribute to fungal self-protection against cyclosporines. Cyclosporine confers on T. inflatum the abilities to outcompete other fungi in competitive interactions and to facilitate fungal infection of insect hosts, which therefore benefits fungal adaptations to different environments.The cycloundecapeptide cyclosporin A (CsA) was first isolated from the insect-pathogenic fungus Tolypocladium inflatum for its antifungal activity and later developed as an immunosuppressant drug. However, the full biosynthetic mechanism of CsA remains unknown and has puzzled researchers for decades. In this study, the biosynthetic gene cluster is suggested to include 12 genes encoding enzymes, including the nonribosomal peptide synthetase (NRPS) (SimA) responsible for assembling the 11 amino acid substrates of cyclosporine and a polyketide synthase (PKS) (SimG) to mediate the production of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-l-threonine (Bmt). Individual deletion of 10 genes, isolation of intermediates, and substrate feeding experiments show that Bmt is biosynthesized by three enzymes, including SimG, SimI, and SimJ. The substrate d-alanine is catalyzed from l-alanine by alanine racemase SimB. Gene cluster transcription is regulated by a putative basic leucine zipper (bZIP)-type protein encoded by the cluster gene SimL. We also found that the cluster cyclophilin (SimC) and transporter (SimD) genes contribute to the tolerance of CsA in the CsA-producing fungus. We also found that cyclosporine production could enable the fungus to outcompete other fungi during cocultivation tests. Deletion of the CsA biosynthetic genes also impaired fungal virulence against insect hosts. Taking all the data together, in addition to proposing a biosynthetic pathway of cyclosporines, the results of this study suggest that CsA produced by this fungus might play important ecological roles in fungal environment interactions
Erik De Clercq - One of the best experts on this subject based on the ideXlab platform.
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inhibition of human immunodeficiency virus type 1 replication in human cells by debio 025 a novel cyclophilin binding agent
Antimicrobial Agents and Chemotherapy, 2008Co-Authors: Roger G Ptak, Philippe Gallay, Dirk Jochmans, Andrew P Halestrap, Urs T Ruegg, Luke A Pallansch, Michael Bobardt, Mariepierre De Bethune, Johan Neyts, Erik De ClercqAbstract:The introduction of highly active antiretroviral therapy has led to significant changes in disease progression and mortality of human immunodeficiency virus (HIV) type 1 (HIV-1) infection and its sequela, AIDS. HIV-1 infection has been turned into a chronic condition that can be treated and that may be manageable over many years (90). However, a significant proportion of patients still fail to have a complete response to treatment over a prolonged time and are at risk of virological rebound, which may lead to the emergence of drug-resistant virus variants (20). The 21 anti-HIV-1 drugs used at present target only three steps in the viral replication cycle, namely, virus fusion, reverse transcription, and the proteolytic processing of viral proteins (22). An inhibitor directed against a fourth step, envelope glycoprotein binding to the CCR5 coreceptor (24, 99), and an inhibitor of a fifth step, integration of viral DNA in the cellular genome (34), were recently approved by FDA. Further inhibitors of the last two steps are in clinical evaluation (23, 86). Thus, the four virus-coded proteins, the envelope glycoprotein, reverse transcriptase (RT), protease, and integrase, are the molecular targets of anti-HIV-1 chemotherapy in the clinic at present. This limits the number of possible combinations that may be used, because cross-resistance of virus strains against drugs targeted to the same viral molecule is common (61). Therefore, novel anti-HIV drugs directed against other steps in the viral replication cycle are highly needed. Cyclophilin A (CypA) was established more than a decade ago to be a valid target in anti-HIV-1 chemotherapy (69, 70). The cellular protein CypA fulfills an essential function early in the HIV-1 replication cycle. It was found to bind specifically to the HIV-1 Gag polyprotein (48). A defined amino acid sequence around G89 and P90 of capsid protein p24 (CA) was identified as the binding site for CypA (17, 31). The affinity of CypA for CA promotes the incorporation of CypA into the virion particles during assembly (10, 30, 87). Experimental evidence indicates that the CypA-CA interaction is essential for HIV-1 replication; inhibition of this interaction impairs HIV-1 replication in human cells (10, 37, 81, 82). The step in the viral replication cycle where CypA is involved was demonstrated to be an event after penetration of the virus particle and before integration of the double-stranded viral DNA into the cellular genome (13, 51, 82). CypA is a member of the immunophilin class of proteins. These ubiquitous cellular proteins possess cis-trans prolyl isomerase (PPIase) activities (27) and are assumed to be involved in protein folding and to function as chaperones in intracellular transport (78). Cyclophilins are also known to be the intracellular receptor molecules for cyclosporines (35), a class of cyclic undecapeptides produced by Trichoderma polysporum (25, 71). Binding of cyclosporines to cyclophilins leads to the blockade of the isomerase activity. The most prominent representative of this class of compounds is cyclosporine (Cs), a potent inhibitor of T-cell activation widely used in the clinic as an immunosuppressant in organ transplantation (11). When Cs is bound to CypA it forms a ternary complex with calcineurin. This binding inhibits the phosphatase activity of calcineurin, which is crucial for signal transduction in the activation cascade of T cells. Inhibition of calcineurin function is thus the molecular basis of the immunosuppressive action of Cs (8). The structure of Cs bound to its ligands has been elucidated: two separate domains in the undecapeptide Cs that are involved in binding to CypA and calcineurin, respectively, can be distinguished (40, 43, 66, 94, 98). The immunosuppressive capacity of Cs can therefore be separated from its affinity to CypA by chemical modification. The anti-HIV-1 activity of Cs was first reported in 1988 (91). Evaluation of this drug and many derivatives for inhibition of HIV-1 replication revealed that nonimmunosuppressive Cs analogs had anti-HIV-1 activities equal to or even superior to those of immunosuppressive analogs (7, 10). A clear correlation was evident, however, between their antiviral activities and the ability of cyclosporines to bind to their cellular receptor protein, CypA. Modification of amino acids in the calcineurin binding domain of Cs, in particular, substitution of the undecapeptide at position 4, was shown to abolish the formation of the ternary complex with calcineurin and, thus, led to the loss of its immunosuppressive capacity (62, 96, 105). Binding to CypA and anti-HIV-1 activity were not impaired by such modifications. (Methyl Ile4)cyclosporine (NIM811), a nonimmunosuppressive cyclosporine with a high affinity for binding to CypA and also potent and selective anti-HIV-1 activity, was characterized in more detail (68). Mode-of-action studies revealed that NIM811 inhibited the CypA-CA interaction in a dose-dependent manner (10, 87) and that an early step in the replication cycle of HIV-1 was impaired in the presence of the compound (10, 51, 82). NIM811 was produced by fermentation of the fungus Tolypocladium niveum, followed by extraction and purification (89). Chemical derivatization procedures for the production of a large variety of Cs derivatives are well established. A large amount of knowledge exists about the structure-activity relationships regarding immunosuppressive capacity, CypA binding, and anti-HIV-1 activity (7, 10, 41, 96, 105). We set out to synthesize a series of novel cyclosporines using Cs as a starting material. The goal was to produce by chemical derivatization nonimmunosuppressive cyclosporines with increased affinities for cyclophilin, which was expected to yield derivatives with improved anti-HIV-1 activities. The most promising compound in this series of derivatives turned out to be (d-MeAla3-EtVal4)cyclosporine (Debio-025; where Me and Et are methyl and ethyl, respectively) (97). The in vitro pharmacological profile of this novel CypA-blocking compound and, in particular, its anti-HIV-1 potential are described in this report.
Xiuqing Yang - One of the best experts on this subject based on the ideXlab platform.
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Cyclosporine biosynthesis in Tolypocladium inflatum benefits fungal adaptation to the environment
mBio, 2018Co-Authors: Xiuqing Yang, Peng Feng, Ying Yin, Kathryn Bushley, Joseph W. Spatafora, Chengshu WangAbstract:The cycloundecapeptide cyclosporin A (CsA) was first isolated from the insect-pathogenic fungus Tolypocladium inflatum for its antifungal activity and later developed as an immunosuppressant drug. However, the full biosynthetic mechanism of CsA remains unknown and has puzzled researchers for decades. In this study, the biosynthetic gene cluster is suggested to include 12 genes encoding enzymes, including the nonribosomal peptide synthetase (NRPS) (SimA) responsible for assembling the 11 amino acid substrates of cyclosporine and a polyketide synthase (PKS) (SimG) to mediate the production of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-l-threonine (Bmt). Individual deletion of 10 genes, isolation of intermediates, and substrate feeding experiments show that Bmt is biosynthesized by three enzymes, including SimG, SimI, and SimJ. The substrate d-alanine is catalyzed from l-alanine by alanine racemase SimB. Gene cluster transcription is regulated by a putative basic leucine zipper (bZIP)-type protein encoded by the cluster gene SimL We also found that the cluster cyclophilin (SimC) and transporter (SimD) genes contribute to the tolerance of CsA in the CsA-producing fungus. We also found that cyclosporine production could enable the fungus to outcompete other fungi during cocultivation tests. Deletion of the CsA biosynthetic genes also impaired fungal virulence against insect hosts. Taking all the data together, in addition to proposing a biosynthetic pathway of cyclosporines, the results of this study suggest that CsA produced by this fungus might play important ecological roles in fungal environment interactions.IMPORTANCE The cyclopeptide cyclosporin A was first isolated from the filamentous fungus Tolypocladium inflatum showing antifungal activity and was later developed as an immunosuppressant drug. We report the biosynthetic mechanism of cyclosporines that are mediated by a cluster of genes encoding NRPS and PKS controlled by a bZIP-type transcriptional regulator. The two unusual amino acids Bmt and d-Ala are produced by the PKS pathway and alanine racemase, respectively. The cyclophilin and transporter genes jointly contribute to fungal self-protection against cyclosporines. Cyclosporine confers on T. inflatum the abilities to outcompete other fungi in competitive interactions and to facilitate fungal infection of insect hosts, which therefore benefits fungal adaptations to different environments.
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Cyclosporine Biosynthesis in Tolypocladium inflatum Benefits Fungal Adaptation to the Environment
American Society for Microbiology, 2018Co-Authors: Xiuqing Yang, Peng Feng, Ying Yin, Kathryn Bushley, Joseph W. Spatafora, Chengshu WangAbstract:The cyclopeptide cyclosporin A was first isolated from the filamentous fungus Tolypocladium inflatum showing antifungal activity and was later developed as an immunosuppressant drug. We report the biosynthetic mechanism of cyclosporines that are mediated by a cluster of genes encoding NRPS and PKS controlled by a bZIP-type transcriptional regulator. The two unusual amino acids Bmt and d-Ala are produced by the PKS pathway and alanine racemase, respectively. The cyclophilin and transporter genes jointly contribute to fungal self-protection against cyclosporines. Cyclosporine confers on T. inflatum the abilities to outcompete other fungi in competitive interactions and to facilitate fungal infection of insect hosts, which therefore benefits fungal adaptations to different environments.The cycloundecapeptide cyclosporin A (CsA) was first isolated from the insect-pathogenic fungus Tolypocladium inflatum for its antifungal activity and later developed as an immunosuppressant drug. However, the full biosynthetic mechanism of CsA remains unknown and has puzzled researchers for decades. In this study, the biosynthetic gene cluster is suggested to include 12 genes encoding enzymes, including the nonribosomal peptide synthetase (NRPS) (SimA) responsible for assembling the 11 amino acid substrates of cyclosporine and a polyketide synthase (PKS) (SimG) to mediate the production of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-l-threonine (Bmt). Individual deletion of 10 genes, isolation of intermediates, and substrate feeding experiments show that Bmt is biosynthesized by three enzymes, including SimG, SimI, and SimJ. The substrate d-alanine is catalyzed from l-alanine by alanine racemase SimB. Gene cluster transcription is regulated by a putative basic leucine zipper (bZIP)-type protein encoded by the cluster gene SimL. We also found that the cluster cyclophilin (SimC) and transporter (SimD) genes contribute to the tolerance of CsA in the CsA-producing fungus. We also found that cyclosporine production could enable the fungus to outcompete other fungi during cocultivation tests. Deletion of the CsA biosynthetic genes also impaired fungal virulence against insect hosts. Taking all the data together, in addition to proposing a biosynthetic pathway of cyclosporines, the results of this study suggest that CsA produced by this fungus might play important ecological roles in fungal environment interactions
Brigitte Rosenwirth - One of the best experts on this subject based on the ideXlab platform.
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Mode of Action of SDZ NIM 811, a Nonimmunosuppressive Cyclosporin A Analog with Activity against Human Immunodeficiency Virus (HIV) Type 1: Interference with HIV Protein-Cyclophilin A Interactions
Journal of virology, 1995Co-Authors: Andreas Billich, F Hammerschmid, P Peichl, Roland Wenger, G Zenke, Valérie F. J. Quesniaux, Brigitte RosenwirthAbstract:Cyclosporins, in particular the nonimmunosuppressive derivative SDZ NIM 811, exhibit potent anti-human immunodeficiency virus type 1 (HIV-1) activity in vitro. SDZ NIM 811 interferes at two stages of the viral replication cycle: (i) translocation of the preintegration complex to the nucleus and (ii) production of infectious virus particles. Immunosuppressive activity is not correlated with anti-HIV-1 activity of Cyclosporins. However, binding to cyclophilin A, the major cellular receptor protein of Cyclosporins, is a prerequisite for HIV inhibition: all structural changes of the cyclosporin A molecule leading to loss of affinity to cyclophilin abolished the antiviral effect. Cyclosporin derivatives did not interact directly with HIV-1 proteins; cyclophilin was the only detectable receptor protein for antivirally active Cyclosporins. There is no evidence that inhibition of HIV occurs via a gain of function of cyclophilin in the presence of Cyclosporins: the complex of cyclophilin A with SDZ NIM 811 does not bind to calcineurin or to any other viral or cellular proteins under conditions in which calcineurin binding to the cyclophilin A-cyclosporin A complex is easily detectable. Thus, the loss of function caused by binding of Cyclosporins to cyclophilin seems to be sufficient for the anti-HIV effect. Cyclophilin A was demonstrated to bind to HIV-1 p24gag, and the formation of complexes was blocked by Cyclosporins with 50% inhibitory concentrations of about 0.7 microM. HIV-2 and simian immunodeficiency virus are only weakly or not at all inhibited by Cyclosporins. For gag-encoded proteins derived from HIV-1, HIV-2, or simian immunodeficiency virus particles, cyclophilin-binding capacity correlated with sensitivity of the viruses to inhibition by Cyclosporins. Cyclophilin A also binds to HIV-1 proteins other than gag-encoded proteins, namely, p17gag, Nef, Vif, and gp120env; the biological significance of these interactions is questionable. We conclude that HIV-1 Gag-cyclophilin A interaction may be essential in HIV-1 replication, and interference with this interaction may be the molecular basis for the antiviral activity of Cyclosporins.
Roger G Ptak - One of the best experts on this subject based on the ideXlab platform.
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inhibition of human immunodeficiency virus type 1 replication in human cells by debio 025 a novel cyclophilin binding agent
Antimicrobial Agents and Chemotherapy, 2008Co-Authors: Roger G Ptak, Philippe Gallay, Dirk Jochmans, Andrew P Halestrap, Urs T Ruegg, Luke A Pallansch, Michael Bobardt, Mariepierre De Bethune, Johan Neyts, Erik De ClercqAbstract:The introduction of highly active antiretroviral therapy has led to significant changes in disease progression and mortality of human immunodeficiency virus (HIV) type 1 (HIV-1) infection and its sequela, AIDS. HIV-1 infection has been turned into a chronic condition that can be treated and that may be manageable over many years (90). However, a significant proportion of patients still fail to have a complete response to treatment over a prolonged time and are at risk of virological rebound, which may lead to the emergence of drug-resistant virus variants (20). The 21 anti-HIV-1 drugs used at present target only three steps in the viral replication cycle, namely, virus fusion, reverse transcription, and the proteolytic processing of viral proteins (22). An inhibitor directed against a fourth step, envelope glycoprotein binding to the CCR5 coreceptor (24, 99), and an inhibitor of a fifth step, integration of viral DNA in the cellular genome (34), were recently approved by FDA. Further inhibitors of the last two steps are in clinical evaluation (23, 86). Thus, the four virus-coded proteins, the envelope glycoprotein, reverse transcriptase (RT), protease, and integrase, are the molecular targets of anti-HIV-1 chemotherapy in the clinic at present. This limits the number of possible combinations that may be used, because cross-resistance of virus strains against drugs targeted to the same viral molecule is common (61). Therefore, novel anti-HIV drugs directed against other steps in the viral replication cycle are highly needed. Cyclophilin A (CypA) was established more than a decade ago to be a valid target in anti-HIV-1 chemotherapy (69, 70). The cellular protein CypA fulfills an essential function early in the HIV-1 replication cycle. It was found to bind specifically to the HIV-1 Gag polyprotein (48). A defined amino acid sequence around G89 and P90 of capsid protein p24 (CA) was identified as the binding site for CypA (17, 31). The affinity of CypA for CA promotes the incorporation of CypA into the virion particles during assembly (10, 30, 87). Experimental evidence indicates that the CypA-CA interaction is essential for HIV-1 replication; inhibition of this interaction impairs HIV-1 replication in human cells (10, 37, 81, 82). The step in the viral replication cycle where CypA is involved was demonstrated to be an event after penetration of the virus particle and before integration of the double-stranded viral DNA into the cellular genome (13, 51, 82). CypA is a member of the immunophilin class of proteins. These ubiquitous cellular proteins possess cis-trans prolyl isomerase (PPIase) activities (27) and are assumed to be involved in protein folding and to function as chaperones in intracellular transport (78). Cyclophilins are also known to be the intracellular receptor molecules for cyclosporines (35), a class of cyclic undecapeptides produced by Trichoderma polysporum (25, 71). Binding of cyclosporines to cyclophilins leads to the blockade of the isomerase activity. The most prominent representative of this class of compounds is cyclosporine (Cs), a potent inhibitor of T-cell activation widely used in the clinic as an immunosuppressant in organ transplantation (11). When Cs is bound to CypA it forms a ternary complex with calcineurin. This binding inhibits the phosphatase activity of calcineurin, which is crucial for signal transduction in the activation cascade of T cells. Inhibition of calcineurin function is thus the molecular basis of the immunosuppressive action of Cs (8). The structure of Cs bound to its ligands has been elucidated: two separate domains in the undecapeptide Cs that are involved in binding to CypA and calcineurin, respectively, can be distinguished (40, 43, 66, 94, 98). The immunosuppressive capacity of Cs can therefore be separated from its affinity to CypA by chemical modification. The anti-HIV-1 activity of Cs was first reported in 1988 (91). Evaluation of this drug and many derivatives for inhibition of HIV-1 replication revealed that nonimmunosuppressive Cs analogs had anti-HIV-1 activities equal to or even superior to those of immunosuppressive analogs (7, 10). A clear correlation was evident, however, between their antiviral activities and the ability of cyclosporines to bind to their cellular receptor protein, CypA. Modification of amino acids in the calcineurin binding domain of Cs, in particular, substitution of the undecapeptide at position 4, was shown to abolish the formation of the ternary complex with calcineurin and, thus, led to the loss of its immunosuppressive capacity (62, 96, 105). Binding to CypA and anti-HIV-1 activity were not impaired by such modifications. (Methyl Ile4)cyclosporine (NIM811), a nonimmunosuppressive cyclosporine with a high affinity for binding to CypA and also potent and selective anti-HIV-1 activity, was characterized in more detail (68). Mode-of-action studies revealed that NIM811 inhibited the CypA-CA interaction in a dose-dependent manner (10, 87) and that an early step in the replication cycle of HIV-1 was impaired in the presence of the compound (10, 51, 82). NIM811 was produced by fermentation of the fungus Tolypocladium niveum, followed by extraction and purification (89). Chemical derivatization procedures for the production of a large variety of Cs derivatives are well established. A large amount of knowledge exists about the structure-activity relationships regarding immunosuppressive capacity, CypA binding, and anti-HIV-1 activity (7, 10, 41, 96, 105). We set out to synthesize a series of novel cyclosporines using Cs as a starting material. The goal was to produce by chemical derivatization nonimmunosuppressive cyclosporines with increased affinities for cyclophilin, which was expected to yield derivatives with improved anti-HIV-1 activities. The most promising compound in this series of derivatives turned out to be (d-MeAla3-EtVal4)cyclosporine (Debio-025; where Me and Et are methyl and ethyl, respectively) (97). The in vitro pharmacological profile of this novel CypA-blocking compound and, in particular, its anti-HIV-1 potential are described in this report.
