18 results on '"Lin, Gang"'
Search Results
2. Macrocyclic Peptides that Selectively Inhibit the Mycobacterium tuberculosis Proteasome.
- Author
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Zhang H, Hsu HC, Kahne SC, Hara R, Zhan W, Jiang X, Burns-Huang K, Ouellette T, Imaeda T, Okamoto R, Kawasaki M, Michino M, Wong TT, Toita A, Yukawa T, Moraca F, Vendome J, Saha P, Sato K, Aso K, Ginn J, Meinke PT, Foley M, Nathan CF, Darwin KH, Li H, and Lin G
- Subjects
- Anti-Bacterial Agents chemistry, Anti-Bacterial Agents pharmacology, Drug Design, Humans, Mycobacterium tuberculosis drug effects, Peptides, Cyclic chemistry, Structure-Activity Relationship, Mycobacterium tuberculosis enzymology, Peptides, Cyclic pharmacology, Proteasome Endopeptidase Complex metabolism, Proteasome Inhibitors chemistry, Proteasome Inhibitors pharmacology
- Abstract
Treatment of tuberculosis (TB) currently takes at least 6 months. Latent Mycobacterium tuberculosis (Mtb) is phenotypically tolerant to most anti-TB drugs. A key hypothesis is that drugs that kill nonreplicating (NR) Mtb may shorten treatment when used in combination with conventional drugs. The Mtb proteasome (Mtb20S) could be such a target because its pharmacological inhibition kills NR Mtb and its genetic deletion renders Mtb unable to persist in mice. Here, we report a series of macrocyclic peptides that potently and selectively target the Mtb20S over human proteasomes, including macrocycle 6 . The cocrystal structure of macrocycle 6 with Mtb20S revealed structural bases for the species selectivity. Inhibition of 20S within Mtb by 6 dose dependently led to the accumulation of Pup-tagged GFP that is degradable but resistant to depupylation and death of nonreplicating Mtb under nitrosative stress. These results suggest that compounds of this class have the potential to develop as anti-TB therapeutics.
- Published
- 2021
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3. Selective Phenylimidazole-Based Inhibitors of the Mycobacterium tuberculosis Proteasome.
- Author
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Zhan W, Hsu HC, Morgan T, Ouellette T, Burns-Huang K, Hara R, Wright AG, Imaeda T, Okamoto R, Sato K, Michino M, Ramjee M, Aso K, Meinke PT, Foley M, Nathan CF, Li H, and Lin G
- Subjects
- Imidazoles chemistry, Microbial Sensitivity Tests, Mycobacterium tuberculosis enzymology, Proteasome Inhibitors chemistry, Reactive Nitrogen Species metabolism, Structure-Activity Relationship, Imidazoles pharmacology, Mycobacterium tuberculosis drug effects, Proteasome Inhibitors pharmacology
- Abstract
Proteasomes of pathogenic microbes have become attractive targets for anti-infectives. Coevolving with its human host, Mycobacterium tuberculosis (Mtb) has developed mechanisms to resist host-imposed nitrosative and oxidative stresses. Genetic deletion or pharmacological inhibition of the Mtb proteasome (Mtb20S) renders nonreplicating Mtb susceptible to reactive nitrogen species in vitro and unable to survive in the lungs of mice, validating the Mtb proteasome as a promising target for anti-Mtb agents. Using a structure-guided and flow chemistry-enabled study of structure-activity relationships, we developed phenylimidazole-based peptidomimetics that are highly potent for Mtb20S. X-ray structures of selected compounds with Mtb20S shed light on their selectivity for mycobacterial over human proteasomes.
- Published
- 2019
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4. Rational Design of Selective and Bioactive Inhibitors of the Mycobacterium tuberculosis Proteasome.
- Author
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Totaro KA, Barthelme D, Simpson PT, Jiang X, Lin G, Nathan CF, Sauer RT, and Sello JK
- Subjects
- Cell Line, Drug Design, Humans, Models, Molecular, Mycobacterium tuberculosis drug effects, Peptides, Cyclic chemistry, Peptides, Cyclic pharmacology, Proteasome Endopeptidase Complex metabolism, Proteasome Inhibitors chemistry, Proteasome Inhibitors pharmacology, Protein Binding, Substrate Specificity, Mycobacterium tuberculosis enzymology, Peptides, Cyclic chemical synthesis, Proteasome Inhibitors chemical synthesis
- Abstract
The 20S core particle of the proteasome in Mycobacterium tuberculosis (Mtb) is a promising, yet unconventional, drug target. This multimeric peptidase is not essential, yet degrades proteins that have become damaged and toxic via reactions with nitric oxide (and/or the associated reactive nitrogen intermediates) produced during the host immune response. Proteasome inhibitors could render Mtb susceptible to the immune system, but they would only be therapeutically viable if they do not inhibit the essential 20S counterpart in humans. Selective inhibitors of the Mtb 20S were designed and synthesized on the bases of both its unique substrate preferences and the structures of substrate-mimicking covalent inhibitors of eukaryotic proteasomes called syringolins. Unlike the parent syringolins, the designed analogues weakly inhibit the human 20S (Hs 20S) proteasome and preferentially inhibit Mtb 20S over the human counterpart by as much as 74-fold. Moreover, they can penetrate the mycobacterial cell envelope and render Mtb susceptible to nitric oxide-mediated stress. Importantly, they do not inhibit the growth of human cell lines in vitro and thus may be starting points for tuberculosis drug development.
- Published
- 2017
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5. Structural Basis for the Species-Selective Binding of N,C-Capped Dipeptides to the Mycobacterium tuberculosis Proteasome.
- Author
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Hsu HC, Singh PK, Fan H, Wang R, Sukenick G, Nathan C, Lin G, and Li H
- Subjects
- Animals, Bacterial Proteins metabolism, Binding Sites, Catalytic Domain, Crystallization, Crystallography, X-Ray, Dipeptides metabolism, Dipeptides pharmacology, Glutamine chemistry, Glutamine metabolism, Humans, Mice, Models, Molecular, Molecular Structure, Proteasome Endopeptidase Complex metabolism, Proteasome Inhibitors chemistry, Proteasome Inhibitors metabolism, Proteasome Inhibitors pharmacology, Protein Binding, Protein Conformation drug effects, Protein Domains, Serine chemistry, Serine metabolism, Species Specificity, Structure-Activity Relationship, Bacterial Proteins chemistry, Dipeptides chemistry, Mycobacterium tuberculosis metabolism, Proteasome Endopeptidase Complex chemistry
- Abstract
The Mycobacterium tuberculosis (Mtb) 20S proteasome is vital for the pathogen to survive under nitrosative stress in vitro and to persist in mice. To qualify for drug development, inhibitors targeting Mtb 20S must spare both the human constitutive proteasome (c-20S) and immunoproteasome (i-20S). We recently reported members of a family of noncovalently binding dipeptide proteasome inhibitors that are highly potent and selective for Mtb 20S over human c-20S and i-20S. To understand the structural basis of their potency and selectivity, we have studied the structure-activity relationship of six derivatives and solved their cocrystal structures with Mtb 20S. The dipeptide inhibitors form an antiparallel β-strand with the active site β-strands. Selectivity is conferred by several features of Mtb 20S relative to its mouse counterparts, including a larger S1 pocket, additional hydrogen bonds in the S3 pocket, and hydrophobic interactions in the S4 pocket. Serine-20 and glutamine-22 of Mtb 20S interact with the dipeptides and confer Mtb-specific inhibition over c-20S and i-20S. The Mtb 20S and mammalian i-20S have a serine-27 that interacts strongly with the dipeptides, potentially explaining the higher inhibitory activity of the dipeptides toward i-20S over c-20S. This detailed structural knowledge will aid in optimizing the dipeptides as anti-tuberculosis drugs.
- Published
- 2017
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6. Stressed mycobacteria use the chaperone ClpB to sequester irreversibly oxidized proteins asymmetrically within and between cells.
- Author
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Vaubourgeix J, Lin G, Dhar N, Chenouard N, Jiang X, Botella H, Lupoli T, Mariani O, Yang G, Ouerfelli O, Unser M, Schnappinger D, McKinney J, and Nathan C
- Subjects
- Animals, Anti-Bacterial Agents toxicity, Endopeptidase Clp genetics, Mice, Microbial Viability drug effects, Mycobacterium tuberculosis growth & development, Mycobacterium tuberculosis physiology, Oxidants toxicity, Oxidation-Reduction, Protein Aggregates, Protein Multimerization, Protein Processing, Post-Translational, Protein Transport, Bacterial Proteins metabolism, Endopeptidase Clp metabolism, Mycobacterium tuberculosis drug effects, Mycobacterium tuberculosis enzymology, Oxidative Stress
- Abstract
Mycobacterium tuberculosis (Mtb) defends itself against host immunity and chemotherapy at several levels, including the repair or degradation of irreversibly oxidized proteins (IOPs). To investigate how Mtb deals with IOPs that can neither be repaired nor degraded, we used new chemical and biochemical probes and improved image analysis algorithms for time-lapse microscopy to reveal a defense against stationary phase stress, oxidants, and antibiotics--the sequestration of IOPs into aggregates in association with the chaperone ClpB, followed by the asymmetric distribution of aggregates within bacteria and between their progeny. Progeny born with minimal IOPs grew faster and better survived a subsequent antibiotic stress than their IOP-burdened sibs. ClpB-deficient Mtb had a marked recovery defect from stationary phase or antibiotic exposure and survived poorly in mice. Treatment of tuberculosis might be assisted by drugs that cripple the pathway by which Mtb buffers, sequesters, and asymmetrically distributes IOPs., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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7. N,C-Capped dipeptides with selectivity for mycobacterial proteasome over human proteasomes: role of S3 and S1 binding pockets.
- Author
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Lin G, Chidawanyika T, Tsu C, Warrier T, Vaubourgeix J, Blackburn C, Gigstad K, Sintchak M, Dick L, and Nathan C
- Subjects
- Binding Sites drug effects, Dipeptides chemistry, Dose-Response Relationship, Drug, Humans, Models, Molecular, Molecular Structure, Mycobacterium tuberculosis chemistry, Structure-Activity Relationship, Dipeptides pharmacology, Mycobacterium tuberculosis drug effects, Proteasome Endopeptidase Complex metabolism
- Abstract
We identified N,C-capped dipeptides that are selective for the Mycobacterium tuberculosis proteasome over human constitutive and immunoproteasomes. Differences in the S3 and S1 binding pockets appeared to account for the species selectivity. The inhibitors can penetrate mycobacteria and kill nonreplicating M. tuberculosis under nitrosative stress.
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- 2013
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8. Fellutamide B is a potent inhibitor of the Mycobacterium tuberculosis proteasome.
- Author
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Lin G, Li D, Chidawanyika T, Nathan C, and Li H
- Subjects
- Bacterial Proteins chemistry, Bacterial Proteins genetics, Catalytic Domain, Drug Evaluation, Preclinical, Humans, In Vitro Techniques, Kinetics, Models, Molecular, Mutant Proteins antagonists & inhibitors, Mutant Proteins chemistry, Mutant Proteins genetics, Mycobacterium tuberculosis genetics, Proteasome Endopeptidase Complex chemistry, Proteasome Endopeptidase Complex genetics, Protein Conformation, Saccharomyces cerevisiae Proteins antagonists & inhibitors, Saccharomyces cerevisiae Proteins chemistry, Species Specificity, Bacterial Proteins antagonists & inhibitors, Lipopeptides pharmacology, Mycobacterium tuberculosis drug effects, Mycobacterium tuberculosis enzymology, Protease Inhibitors pharmacology, Proteasome Inhibitors
- Abstract
Via high-throughput screening of a natural compound library, we have identified a lipopeptide aldehyde, fellutamide B (1), as the most potent inhibitor of the Mycobacterium tuberculosis (Mtb) proteasome tested to date. Kinetic studies reveal that 1 inhibits both Mtb and human proteasomes in a time-dependent manner under steady-state condition. Remarkably, 1 inhibits the Mtb proteasome in a single-step binding mechanism with K(i)=6.8 nM, whereas it inhibits the human proteasome beta5 active site following a two-step mechanism with K(i)=11.5 nM and K(i)(*)=0.93 nM. Co-crystallization of 1 bound to the Mtb proteasome revealed a structural basis for the tight binding of 1 to the active sites of the Mtb proteasome. The hemiacetal group of 1 in the Mtb proteasome takes the (R)-configuration, whereas in the yeast proteasome it takes the (S)-configuration, indicating that the pre-chiral CHO group of 1 binds to the active site Thr1 in a different orientation. Re-examination of the structure of the yeast proteasome in complex with 1 showed significant conformational changes at the substrate-binding cleft along the active site. These structural differences are consistent with the different kinetic mechanisms of 1 against Mtb and human proteasomes., (Copyright 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
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9. Structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20S proteasome.
- Author
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Li D, Li H, Wang T, Pan H, Lin G, and Li H
- Subjects
- Cryoelectron Microscopy, Crystallography, X-Ray, Models, Molecular, Protein Conformation, Protein Structure, Quaternary, Mycobacterium tuberculosis chemistry, Mycobacterium tuberculosis enzymology, Proteasome Endopeptidase Complex chemistry, Proteasome Endopeptidase Complex metabolism, Protein Multimerization
- Abstract
Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin-proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo-electron microscopy and X-ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the beta-subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven alpha-subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.
- Published
- 2010
- Full Text
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10. Structural insights on the Mycobacterium tuberculosis proteasomal ATPase Mpa.
- Author
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Wang T, Li H, Lin G, Tang C, Li D, Nathan C, Darwin KH, and Li H
- Subjects
- Adenosine Triphosphatases genetics, Adenosine Triphosphatases metabolism, Amino Acid Sequence, Crystallography, X-Ray, Models, Molecular, Molecular Sequence Data, Mycobacterium tuberculosis metabolism, Protein Conformation, Protein Folding, Adenosine Triphosphatases chemistry, Mycobacterium tuberculosis enzymology, Proteasome Endopeptidase Complex metabolism
- Abstract
Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPgammaS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved interdomain showed a five stranded double beta barrel structure containing a Greek key motif. Structure and mutational analysis indicate a major role of the interdomain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome.
- Published
- 2009
- Full Text
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11. Nitazoxanide kills replicating and nonreplicating Mycobacterium tuberculosis and evades resistance.
- Author
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de Carvalho LP, Lin G, Jiang X, and Nathan C
- Subjects
- Antiparasitic Agents, Antitubercular Agents pharmacology, Dose-Response Relationship, Drug, Drug Resistance, Microbial Sensitivity Tests, Mycobacterium tuberculosis growth & development, Nitro Compounds, Mycobacterium tuberculosis drug effects, Thiazoles pharmacology
- Abstract
We report here that nitazoxanide (NTZ) and its active metabolite kill replicating and nonreplicating M. tuberculosis at low microg/mL levels. NTZ appears to evade resistance, as we were unable to recover resistant colonies, using up to 10(12) colony forming units. Therefore, NTZ is a novel lead compound that kills replicating and nonreplicating M. tuberculosis by a novel mechanism of action, which appears to bypass the development of resistance.
- Published
- 2009
- Full Text
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12. Inhibitors selective for mycobacterial versus human proteasomes.
- Author
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Lin G, Li D, de Carvalho LP, Deng H, Tao H, Vogt G, Wu K, Schneider J, Chidawanyika T, Warren JD, Li H, and Nathan C
- Subjects
- Catalytic Domain drug effects, Humans, Hydrogen Bonding, Kinetics, Models, Molecular, Mycobacterium tuberculosis growth & development, Oxazolidinones metabolism, Oxazolidinones pharmacology, Protease Inhibitors chemistry, Proteasome Endopeptidase Complex chemistry, Proteasome Endopeptidase Complex metabolism, Protein Carbonylation drug effects, Protein Conformation drug effects, Protein Subunits, Substrate Specificity, Thiazoles pharmacology, Threonine metabolism, Mycobacterium tuberculosis drug effects, Mycobacterium tuberculosis enzymology, Protease Inhibitors pharmacology, Proteasome Inhibitors
- Abstract
Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis. Given that the proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-one compounds kill non-replicating M. tuberculosis and act as selective suicide-substrate inhibitors of the M. tuberculosis proteasome by cyclocarbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose hydrogen bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-one compounds to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homologue.
- Published
- 2009
- Full Text
- View/download PDF
13. Distinct specificities of Mycobacterium tuberculosis and mammalian proteasomes for N-acetyl tripeptide substrates.
- Author
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Lin G, Tsu C, Dick L, Zhou XK, and Nathan C
- Subjects
- Amino Acids chemistry, Animals, Boronic Acids pharmacology, Bortezomib, Cattle, Humans, Inhibitory Concentration 50, Kinetics, Mutation, Mycobacterium tuberculosis chemistry, Oligopeptides chemistry, Proteasome Endopeptidase Complex chemistry, Protein Binding, Pyrazines pharmacology, Rhodococcus metabolism, Substrate Specificity, Mycobacterium tuberculosis metabolism, Peptides chemistry
- Abstract
The proteasome of Mycobacterium tuberculosis (Mtb) is a validated and drug-treatable target for therapeutics. To lay ground-work for developing peptide-based inhibitors with a useful degree of selectivity for the Mtb proteasome over those of the host, we used a library of 5,920 N-acetyl tripeptide-aminomethylcoumarins to contrast the substrate preferences of the recombinant Mtb proteasome wild type and open gate mutant, the Rhodococcus erythropolis proteasome, and the bovine proteasome with activator PA28. The Mtb proteasome was distinctive in strictly preferring P1 = tryptophan, particularly in combination with P3 = glycine, proline, lysine or arginine. Screening results were validated with Michalis-Menten kinetic analyses of 21 oligopeptide aminomethyl-coumarin substrates. Bortezomib, a proteasome inhibitor in clinical use, and 17 analogs varying only at P1 were used to examine the differential impact of inhibitors on human and Mtb proteasomes. The results with the inhibitor panel confirmed those with the substrate panel in demonstrating differential preferences of Mtb and mammalian proteasomes at the P1 amino acid. Changing P1 in bortezomib from Leu to m-CF(3)-Phe led to a 220-fold increase in IC(50) against the human proteasome, whereas changing a P1 Ala to m-F-Phe decreased the IC(50) 400-fold against the Mtb proteasome. The change of a P1 Ala to m-Cl-Phe led to an 8000-fold shift in inhibitory potency in favor of the Mtb proteasome, resulting in 8-fold selectivity. Combinations of preferred amino acids at different sites may thus improve the species selectivity of peptide-based inhibitors that target the Mtb proteasome.
- Published
- 2008
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14. Selective killing of nonreplicating mycobacteria.
- Author
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Bryk R, Gold B, Venugopal A, Singh J, Samy R, Pupek K, Cao H, Popescu C, Gurney M, Hotha S, Cherian J, Rhee K, Ly L, Converse PJ, Ehrt S, Vandal O, Jiang X, Schneider J, Lin G, and Nathan C
- Subjects
- Acyltransferases genetics, Animals, Bacterial Proteins genetics, Cells, Cultured, Colony Count, Microbial, Enzyme Inhibitors pharmacology, Gene Deletion, Genetic Complementation Test, Guinea Pigs, Hypoxia immunology, Lung microbiology, Macrophages drug effects, Macrophages microbiology, Molecular Structure, Mycobacterium tuberculosis genetics, Mycobacterium tuberculosis pathogenicity, Nitric Oxide immunology, Rhodanine chemistry, Rhodanine toxicity, Tuberculosis immunology, Tuberculosis microbiology, Virulence, Virulence Factors antagonists & inhibitors, Virulence Factors genetics, Acyltransferases antagonists & inhibitors, Antitubercular Agents pharmacology, Bacterial Proteins antagonists & inhibitors, Microbial Viability drug effects, Mycobacterium tuberculosis drug effects, Mycobacterium tuberculosis immunology, Rhodanine pharmacology
- Abstract
Antibiotics are typically more effective against replicating rather than nonreplicating bacteria. However, a major need in global health is to eradicate persistent or nonreplicating subpopulations of bacteria such as Mycobacterium tuberculosis (Mtb). Hence, identifying chemical inhibitors that selectively kill bacteria that are not replicating is of practical importance. To address this, we screened for inhibitors of dihydrolipoamide acyltransferase (DlaT), an enzyme required by Mtb to cause tuberculosis in guinea pigs and used by the bacterium to resist nitric oxide-derived reactive nitrogen intermediates, a stress encountered in the host. Chemical screening for inhibitors of Mtb DlaT identified select rhodanines as compounds that almost exclusively kill nonreplicating mycobacteria in synergy with products of host immunity, such as nitric oxide and hypoxia, and are effective on bacteria within macrophages, a cellular reservoir for latent Mtb. Compounds that kill nonreplicating pathogens in cooperation with host immunity could complement the conventional chemotherapy of infectious disease.
- Published
- 2008
- Full Text
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15. Mycobacterium tuberculosis prcBA genes encode a gated proteasome with broad oligopeptide specificity.
- Author
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Lin G, Hu G, Tsu C, Kunes YZ, Li H, Dick L, Parsons T, Li P, Chen Z, Zwickl P, Weich N, and Nathan C
- Subjects
- Bacterial Proteins metabolism, Binding Sites, Gene Expression Regulation, Bacterial, Ion Channel Gating, Microscopy, Electron, Mutation, Mycobacterium tuberculosis drug effects, Mycobacterium tuberculosis metabolism, Protease Inhibitors pharmacology, Proteasome Endopeptidase Complex chemistry, Proteasome Inhibitors, Substrate Specificity, Subtilisins metabolism, Titrimetry, Bacterial Proteins genetics, Mycobacterium tuberculosis genetics, Oligopeptides metabolism, Proteasome Endopeptidase Complex genetics, Proteasome Endopeptidase Complex metabolism, Subtilisins genetics
- Abstract
Genes predicted to be associated with the putative proteasome of Mycobacterium tuberculosis (Mtb) play a critical role in defence of the bacillus against nitrosative stress. However, proteasomes are uncommon in eubacteria and it remains to be established whether Mtb's prcBA genes in fact encode a proteasome. We found that coexpression of recombinant PrcB and PrcA in Escherichia coli over a prolonged period at 37 degrees C allowed formation of an alpha(7)beta(7)beta(7)alpha(7), 750 kDa cylindrical stack of four rings in which all 14 beta-subunits were proteolytically processed to expose the active site threonine. In contrast to another Actinomycete, Rhodococcus erythropolis, Mtb's beta-chain propeptide was not required for particle assembly. Peptidolytic activity of the 750 kDa particle towards a hydrophobic oligopeptide was nearly two orders of magnitude less than that of the Rhodococcus 20S proteasome, and unlike eukaryotic and archaeal proteasomes, activity of the Mtb 750 kDa particle could not be stimulated by SDS, Mg(2+) or Ca(2+). Electron microscopy revealed what appeared to be obstructed alpha-rings in the Mtb 750 kDa particle. Deletion of the N-terminal octapeptide from Mtb's alpha-chain led to disappearance of the apparent obstruction and a marked increase of peptidolytic activity. Unlike proteasomes isolated from other Actinomycetes, the open-gate Mtb mutant 750 kDa particle cleaved oligopeptides not only after hydrophobic residues but also after basic, acidic and small, neutral amino acids. Thus, Mtb encodes a broadly active, gated proteasome that may work in concert with an endogenous activator.
- Published
- 2006
- Full Text
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16. Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate.
- Author
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Hu G, Lin G, Wang M, Dick L, Xu RM, Nathan C, and Li H
- Subjects
- Binding Sites, Boronic Acids chemistry, Bortezomib, Crystallography, X-Ray, Dipeptides chemistry, Molecular Structure, Protease Inhibitors chemistry, Proteasome Endopeptidase Complex metabolism, Protein Subunits, Pyrazines chemistry, Substrate Specificity, Boronic Acids pharmacology, Dipeptides pharmacology, Mycobacterium tuberculosis enzymology, Proteasome Endopeptidase Complex chemistry, Proteasome Inhibitors
- Abstract
Mycobacterium tuberculosis (Mtb) has the remarkable ability to resist killing by human macrophages. The 750 kDa proteasome, not available in most eubacteria except Actinomycetes, appears to contribute to Mtb's resistance. The crystal structure of the Mtb proteasome at 3.0 A resolution reveals a substrate-binding pocket with composite features of the distinct beta1, beta2 and beta5 substrate binding sites of eukaryotic proteasomes, accounting for the broad specificity of the Mtb proteasome towards oligopeptides described in the companion article [Lin et al. (2006), Mol Microbiol doi:10.1111/j.1365-2958.2005.05035.x]. The substrate entrance at the end of the cylindrical proteasome appears open in the crystal structure due to partial disorder of the alpha-subunit N-terminal residues. However, cryo-electron microscopy of the core particle reveals a closed end, compatible with the density observed in negative-staining electron microscopy that depended on the presence of the N-terminal octapetides of the alpha-subunits in the companion article, suggesting that the Mtb proteasome has a gated structure. We determine for the first time the proteasomal inhibition mechanism of the dipeptidyl boronate N-(4-morpholine)carbonyl-beta-(1-naphthyl)-L-alanine-L-leucine boronic acid (MLN-273), an analogue of the antimyeloma drug bortezomib. The structure improves prospects for designing Mtb-specific proteasomal inhibitors as a novel approach to chemotherapy of tuberculosis.
- Published
- 2006
- Full Text
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17. Characterization of a Mycobacterium tuberculosis proteasomal ATPase homologue.
- Author
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Darwin KH, Lin G, Chen Z, Li H, and Nathan CF
- Subjects
- Adenosine Triphosphatases chemistry, Adenosine Triphosphatases genetics, Animals, Cryoelectron Microscopy, Humans, Mice, Mice, Inbred BALB C, Mycobacterium tuberculosis genetics, Point Mutation, Proteasome Endopeptidase Complex genetics, Proteasome Endopeptidase Complex metabolism, Protein Subunits chemistry, Protein Subunits genetics, Protein Subunits metabolism, Tuberculosis, Pulmonary microbiology, Tuberculosis, Pulmonary mortality, Tuberculosis, Pulmonary physiopathology, Adenosine Triphosphatases metabolism, Mycobacterium tuberculosis drug effects, Mycobacterium tuberculosis enzymology, Mycobacterium tuberculosis pathogenicity, Reactive Nitrogen Species pharmacology
- Abstract
A screen for Mycobacterium tuberculosis (Mtb) mutants sensitive to reactive nitrogen intermediates identified transposon insertions in the presumptive proteasomal ATPase gene mpa (mycobacterium proteasome ATPase; Rv2115c). mpa mutants are attenuated in both wild type and nitric oxide synthase 2 deficient mice. In this work, we show that attenuation of mpa mutants is severe, and that Mpa is an ATPase associated with various cellular activities (AAA) ATPase that forms hexameric rings resembling the eukaryotic complex p97/valosin-containing protein (VCP). Point mutations in the conserved Walker box ATPase motifs of Mpa greatly reduced or abolished ATPase activity in vitro and abrogated protection of Mtb against acidified nitrite. A mutant Mpa protein missing only its last two amino acids retained ATPase activity, yet failed to protect Mtb against nitrite. The corresponding strain was attenuated in mice. Thus, Mpa is an ATPase whose enzymatic activity is necessary but not sufficient to protect against reactive nitrogen intermediates.
- Published
- 2005
- Full Text
- View/download PDF
18. Microbial proteasomes as drug targets.
- Author
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Zhang, Hao and Lin, Gang
- Subjects
- *
DRUG target , *PROTEASOMES , *CHAGAS' disease , *PROTEASOME inhibitors , *MYCOBACTERIUM tuberculosis , *MULTIPLE myeloma , *PARASITES , *PLASMODIUM - Abstract
Proteasomes are compartmentalized, ATP-dependent, N-terminal nucleophile hydrolases that play essentials roles in intracellular protein turnover. They are present in all 3 kingdoms. Pharmacological inhibition of proteasomes is detrimental to cell viability. Proteasome inhibitor rugs revolutionize the treatment of multiple myeloma. Proteasomes in pathogenic microbes such as Mycobacterium tuberculosis (Mtb), Plasmodium falciparum (Pf), and other parasites and worms have been validated as therapeutic targets. Starting with Mtb proteasome, efforts in developing inhibitors selective for microbial proteasomes have made great progress lately. In this review, we describe the strategies and pharmacophores that have been used in developing proteasome inhibitors with potency and selectivity that spare human proteasomes and highlight the development of clinical proteasome inhibitor candidates for treatment of leishmaniasis and Chagas disease. Finally, we discuss the future challenges and therapeutical potentials of the microbial proteasome inhibitors. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
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