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Proceedings of the National Academy of... Apr 2022Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are...
Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. Loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2′-O-methylations within two structurally distinct contexts. Here, we report the structure of a mycobacterial 50S subunit-TlyA complex trapped in a postcatalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. Together with complementary functional analyses, this structure reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144, which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. Base flipping may thus be a common strategy among rRNA methyltransferase enzymes, even in cases where the target site is accessible without such structural reorganization. Finally, functional studies with 30S subunit suggest that the same TlyA interaction surface is employed to recognize this second substrate, but with distinct dependencies on essential conserved residues.
Topics: Bacterial Proteins; Catalytic Domain; Drug Resistance, Bacterial; Methyltransferases; Mycobacterium tuberculosis; Protein Conformation, alpha-Helical; RNA, Ribosomal, 16S; RNA, Ribosomal, 23S; Ribosome Subunits, Large, Bacterial
PubMed: 35357969
DOI: 10.1073/pnas.2120352119 -
Journal of Biomolecular Structure &... Feb 20223CL is the main protease of the novel coronavirus (SARS-CoV-2) responsible for their intracellular duplication. Based on virtual screening technology and molecular...
3CL is the main protease of the novel coronavirus (SARS-CoV-2) responsible for their intracellular duplication. Based on virtual screening technology and molecular dynamics simulation, we found 23 approved clinical drugs such as Viomycin, Capastat, Carfilzomib and Saquinavir, which showed high affinity with the 3CL active sites. These findings showed that there were potential drugs that inhibit SARS-Cov-2's 3CL in the current clinical drug library, and these drugs can be further tested or chemically modified for the treatment of COVID-19.Communicated by Ramaswamy H. Sarma.
Topics: COVID-19; Humans; Molecular Docking Simulation; Peptide Hydrolases; Pharmaceutical Preparations; Protease Inhibitors; SARS-CoV-2
PubMed: 32909528
DOI: 10.1080/07391102.2020.1817786 -
Acta Crystallographica. Section F,... Jul 2023L-2,3-Diaminopropionic acid (L-Dap) is a nonproteinogenic amino acid that plays as an important role as a building block in the biosynthesis of several natural products,...
L-2,3-Diaminopropionic acid (L-Dap) is a nonproteinogenic amino acid that plays as an important role as a building block in the biosynthesis of several natural products, including capreomycin, viomycin, zwittermicin, staphyloferrin and dapdiamide. A previous study reported that CmnB and CmnK are two enzymes that are involved in the formation of L-Dap in the biosynthesis of capreomycin. CmnB catalyzes the condensation reaction of O-phospho-L-serine and L-glutamic acid to generate N-(1-amino-1-carboxyl-2-ethyl)glutamic acid, which subsequently undergoes oxidative hydrolysis via CmnK to generate the product L-Dap. Here, the crystal structure of CmnB in complex with the reaction intermediate PLP-α-aminoacrylate is reported at 2.2 Å resolution. Notably, CmnB is the second known example of a PLP-dependent enzyme that forms a monomeric structure in crystal packing. The crystal structure of CmnB also provides insights into the catalytic mechanism of the enzyme and supports the biosynthetic pathway of L-Dap reported in previous studies.
Topics: Amino Acids; Capreomycin; Crystallography, X-Ray; beta-Alanine; Glutamic Acid
PubMed: 37405487
DOI: 10.1107/S2053230X23005769 -
Non-proteinogenic amino acid based supramolecular hydrogel material for enhanced cell proliferation.Colloids and Surfaces. B, Biointerfaces Jan 2020Supramolecular gel material built from low-molecular-weight (LMW) gelators finds potential applications in various fields, especially in drug delivery, cell...
Supramolecular gel material built from low-molecular-weight (LMW) gelators finds potential applications in various fields, especially in drug delivery, cell encapsulation and delivery, and tissue engineering. The majority of the LMW gelators in these applications are based on functionalized peptides/amino acids consisting of proteinogenic amino acids which are proteolytically unstable. Herein, we have developed a new LMW gelator containing non-proteinogenic amino acid namely 2,3-diaminopropionic acid (Dap), a key precursor in the synthesis of many antibiotics namely viomycin and capreomycin, by functionalizing with fluorenylmethoxycarbonyl at both amino terminals of Dap [Fm-Dap(Fm)]. Hydrogelation test at different pH indicates that Fm-Dap(Fm) can form a hydrogel in a wide range of pH (4.9 to 9.1) with minimum hydrogelation concentration depends on the pH. The mechanical strength and thermal stability of the Fm-Dap(Fm) hydrogel material are found to decrease with increasing pH (acidic > neural/physiological > basic). The thermal stability of Fm-Dap(Fm) hydrogels is pH-dependent and elicits high stability at acidic pH. Also, Fm-Dap(Fm) hydrogels exhibit strong thixotropic property where regelation (self-healing) occurs upon release of stress. Morphological analysis indicates the formation of fibrils, which are entangled to form three dimensional network structures. Several spectroscopic measurements provided evidence for the self-assembly of Fm-Dap(Fm) molecules through intermolecular aromatic π-π stacking and hydrogen bonding interactions during hydrogelation. Interestingly, Fm-Dap(Fm) not only exhibits hydrogel formation but also shows cell viability and enhanced cell proliferation at physiological pH (7.4). Further, Fm-Dap(Fm) forms a hydrogel upon co-incubation with vitamin B and also exhibits release of vitamin B over a period. The current study thus demonstrates the development of a new hydrogel material, based on LMW gelator containing the non-proteinogenic amino acid, which can elicit cell viability, enhanced cell proliferation, drug encapsulation, and drug release properties. Hence, Fm-Dap(Fm) hydrogel could be an ideal material for biomedical applications, especially in tissue engineering and drug delivery.
Topics: Amino Acids; Animals; Cell Proliferation; Drug Delivery Systems; Drug Liberation; Hydrogels; Hydrogen-Ion Concentration; Mice; Models, Molecular; NIH 3T3 Cells; Vitamin B 12; beta-Alanine
PubMed: 31677412
DOI: 10.1016/j.colsurfb.2019.110581 -
Journal of Biomolecular Structure &... Jul 2021The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) which was first reported in Wuhan province of China, has become a deadly pandemic causing alarmingly...
The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) which was first reported in Wuhan province of China, has become a deadly pandemic causing alarmingly high morbidity and mortality. In the absence of new targeted drugs and vaccines against SARS-CoV-2 at present, the choices for effective treatments are limited. Therefore, considering the exigency of the situation, we focused on identifying the available approved drugs as potential inhibitor against the promising Coronavirus drug target, the Main Protease, using computer-aided methods. We created a library of U. S. Food and Drug Administration approved anti-microbial drugs and virtually screened it against the available crystal structures of Main Protease of the virus. The study revealed that Viomycin showed the highest -CDocker energy after docking at the active site of SARS-CoV-2 Main Protease. It is noteworthy that Viomycin showed higher -CDocker energy as compared to the drugs currently under clinical trial for SARS-CoV-2 treatment Ritonavir and Lopinavir. Additionally, Viomycin formed higher number of H-bonds with SARS-CoV-2 Main Protease than its co-crystallised inhibitor compound N3. Molecular dynamics simulation further showed that Viomycin embedded deeply inside the binding pocket and formed robust binding with SARS-CoV-2 Main Protease. Therefore, we propose that Viomycin may act as a potential inhibitor of the Main Protease of SARS-CoV-2. Further optimisations with the drug may support the much-needed rapid response to mitigate the pandemic.Communicated by Ramaswamy H. Sarma.
Topics: Antiviral Agents; Coronavirus 3C Proteases; Drug Repositioning; Molecular Docking Simulation; Protease Inhibitors; SARS-CoV-2; Viomycin
PubMed: 32406317
DOI: 10.1080/07391102.2020.1768902 -
American Journal of TherapeuticsAdvances in drug therapy for pulmonary tuberculosis have had an extraordinary impact on the incidence of tuberculosis in the United States in the past century, which has...
BACKGROUND
Advances in drug therapy for pulmonary tuberculosis have had an extraordinary impact on the incidence of tuberculosis in the United States in the past century, which has decreased from 113/100,000 persons in 1920 to 2.2/100,000 in 2020. Modern treatments have contributed to a remarkable decrease in hospitalizations and mortality and have had a significant impact on the duration and severity of illness, quality of life, and work potential of affected persons.
STUDY QUESTION
What are the milestones of the changes in the expert approach to the pharmacological management of pulmonary tuberculosis in the past century?
STUDY DESIGN
To determine the changes in the experts' approach to the management of pulmonary tuberculosis, as presented in a widely used textbook in the United States.
DATA SOURCES
The chapters describing the management of pulmonary tuberculosis in the 26 editions of Cecil Textbook of Medicine published from 1927 through 2020.
RESULTS
In the preantibiotic era (1927-1943), the Cecil authors emphasized rest, good food, and fresh air as the treatment pillars for pulmonary tuberculosis. The modern era (1947-1971) recorded the discovery of all the drugs that are still used for the initial treatment, in the following order: streptomycin, para-aminosalicylic acid, isoniazid, pyrazinamide, ethambutol, cycloserine, kanamycin, ethionamide, capreomycin, and rifampin. In the postmodern era (1975-2020), therapeutic advances continued with trials of many drug combinations aimed at ameliorating the duration of treatment, drug resistance adverse effects, and poor the recent addition of fluoroquinolones, bedaquiline, and clofazimine.
CONCLUSIONS
The pharmacological management of tuberculosis has remained archaic until the middle of the 20th century. Fundamental progress occurred in a very short period (1947-1971) and was because of the recognition of the antituberculous effect of many antibiotics and chemotherapy agents. The challenges created by mycobacterial infections resistant to multiple drugs remain and have prompted the addition of new drugs in the past decade.
Topics: Humans; Expert Testimony; Quality of Life; Aminosalicylic Acids; Drug Resistance; Drug Resistance, Microbial; Viomycin; Tuberculosis, Pulmonary; Tuberculosis; Streptomycin; Pyrazinamide; Isoniazid; Antitubercular Agents
PubMed: 36301538
DOI: 10.1097/MJT.0000000000001575 -
Frontiers in Chemistry 2022CmnC is an α-ketoglutarate (α-KG)-dependent non-heme iron oxygenase involved in the formation of the l-capreomycidine (l-Cap) moiety in capreomycin (CMN) biosynthesis....
Crystal structure of the α-ketoglutarate-dependent non-heme iron oxygenase CmnC in capreomycin biosynthesis and its engineering to catalyze hydroxylation of the substrate enantiomer.
CmnC is an α-ketoglutarate (α-KG)-dependent non-heme iron oxygenase involved in the formation of the l-capreomycidine (l-Cap) moiety in capreomycin (CMN) biosynthesis. CmnC and its homologues, VioC in viomycin (VIO) biosynthesis and OrfP in streptothricin (STT) biosynthesis, catalyze hydroxylation of l-Arg to form β-hydroxy l-Arg (CmnC and VioC) or β,γ-dihydroxy l-Arg (OrfP). In this study, a combination of biochemical characterization and structural determination was performed to understand the substrate binding environment and substrate specificity of CmnC. Interestingly, despite having a high conservation of the substrate binding environment among CmnC, VioC, and OrfP, only OrfP can hydroxylate the substrate enantiomer d-Arg. Superposition of the structures of CmnC, VioC, and OrfP revealed a similar folds and overall structures. The active site residues of CmnC, VioC, and OrfP are almost conserved; however Leu136, Ser138, and Asp249 around the substrate binding pocket in CmnC are replaced by Gln, Gly, and Tyr in OrfP, respectively. These residues may play important roles for the substrate binding. The mutagenesis analysis revealed that the triple mutant CmnC switches the substrate stereoselectivity from l-Arg to d-Arg with ∼6% relative activity. The crystal structure of CmnC in complex with d-Arg revealed that the substrate loses partial interactions and adopts a different orientation in the binding site. This study provides insights into the enzyme engineering to α-KG non-heme iron oxygenases for adjustment to the substrate stereoselectivity and development of biocatalysts.
PubMed: 36176888
DOI: 10.3389/fchem.2022.1001311 -
The Journal of Physical Chemistry. A Mar 2021The viomycin biosynthesis enzyme VioC is a nonheme iron and α-ketoglutarate-dependent dioxygenase involved in the selective hydroxylation of l-arginine at the...
How Do Electrostatic Perturbations of the Protein Affect the Bifurcation Pathways of Substrate Hydroxylation versus Desaturation in the Nonheme Iron-Dependent Viomycin Biosynthesis Enzyme?
The viomycin biosynthesis enzyme VioC is a nonheme iron and α-ketoglutarate-dependent dioxygenase involved in the selective hydroxylation of l-arginine at the C-position for antibiotics biosynthesis. Interestingly, experimental studies showed that using the substrate analogue, namely, l-homo-arginine, a mixture of products was obtained originating from C-hydroxylation, C-hydroxylation, and C-C-desaturation. To understand how the addition of one CH group to a substrate can lead to such a dramatic change in selectivity and activity, we decided to perform a computational study using quantum mechanical (QM) cluster models. We set up a large active-site cluster model of 245 atoms that includes the oxidant with its first- and second-coordination sphere influences as well as the substrate binding pocket. The model was validated against experimental work from the literature on related enzymes and previous computational studies. Thereafter, possible pathways leading to products and byproducts were investigated for a model containing l-Arg and one for l-homo-Arg as substrate. The calculated free energies of activation predict product distributions that match the experimental observation and give a low-energy C-hydroxylation pathway for l-Arg, while for l-homo-Arg, several barriers are found to be close in energy leading to a mixture of products. We then analyzed the origins of the differences in product distributions using thermochemical, valence bond, and electrostatic models. Our studies show that the C-H and C-H bond strengths of l-Arg and l-homo-Arg are similar; however, external perturbations from an induced electric field of the protein affect the relative C-H bond strengths of l-Arg dramatically and make the C-H bond the weakest and guide the reaction to a selective C-hydroxylation channel. Therefore, the charge distribution in the protein and the induced electric dipole field of the active site of VioC guides the l-Arg substrate activation to C-hydroxylation and disfavors the C-hydroxylation pathway, while this does not occur for l-homo-Arg. Tight substrate positioning and electrostatic perturbations from the second-coordination sphere residues in VioC also result in a slower overall reaction for l-Arg; however, they enable a high substrate selectivity. Our studies highlight the importance of the second-coordination sphere in proteins that position the substrate and oxidant, perturb charge distributions, and enable substrate selectivity.
Topics: Bacterial Proteins; Catalytic Domain; Hydroxylation; Iron; Models, Molecular; Nonheme Iron Proteins; Oxygenases; Static Electricity; Viomycin
PubMed: 33620220
DOI: 10.1021/acs.jpca.1c00141 -
Archives of Microbiology Mar 2023Actinobacteria are the largest bacteria group with 18 significant lineages, which are ubiquitously distributed in all the possible terrains. They are known to produce...
Actinobacteria are the largest bacteria group with 18 significant lineages, which are ubiquitously distributed in all the possible terrains. They are known to produce more than 10,000 medically relevant compounds. Despite their ability to make critical secondary metabolites and genome sequences' availability, these two have not been linked with certainty. With this intent, our study aims at understanding the biosynthetic capacity in terms of secondary metabolite production in 528 Actinobacteria species from five different habitats, viz., soil, water, plants, animals, and humans. In our analysis of 9,646 clusters of 59 different classes, we have documented 64,000 SMs, of which more than 74% were of unique type, while 19% were partially conserved and 7% were conserved compounds. In the case of conserved compounds, we found the highest distribution in soil, 79.12%. We found alternate sources of antibiotics, such as viomycin, vancomycin, teicoplanin, fosfomycin, ficellomycin and patulin, and antitumour compounds, such as doxorubicin and tacrolimus in the soil. Also our study reported alternate sources for the toxin cyanobactin in water and plant isolates. We further analysed the clusters to determine their regulatory pathways and reported the prominent presence of the two component system of TetR/AcrR family, as well as other partial domains like CitB superfamily and HTH superfamily, and discussed their role in secondary metabolite production. This information will be helpful in exploring Actinobacteria from other environments and in discovering new chemical moieties of clinical significance.
Topics: Humans; Animals; Actinobacteria; Bacteria; Genome, Bacterial; Anti-Bacterial Agents; Secondary Metabolism; Multigene Family
PubMed: 36944761
DOI: 10.1007/s00203-023-03482-3 -
ACS Omega Apr 2024Colorectal cancer (CRC) has witnessed a concerning increase in incidence and poses a significant therapeutic challenge due to its poor prognosis. There is a pressing...
Colorectal cancer (CRC) has witnessed a concerning increase in incidence and poses a significant therapeutic challenge due to its poor prognosis. There is a pressing demand to identify novel drug therapies to combat CRC. In this study, we addressed this need by utilizing the pharmacological profiles of anticancer drugs from the Genomics of Drug Sensitivity in Cancer (GDSC) database and developed QSAR models using the Support Vector Machine (SVM) algorithm for prediction of alternative and promiscuous anticancer compounds for CRC treatment. Our QSAR models demonstrated their robustness by achieving a high correlation of determination () after 10-fold cross-validation. For 12 CRC cell lines, ranged from 0.609 to 0.827. The highest performance was achieved for SW1417 and GP5d cell lines with values of 0.827 and 0.786, respectively. Further, we listed the most common chemical descriptors in the drug profiles of the CRC cell lines and we also further reported the correlation of these descriptors with drug activity. The KRFP314 fingerprint was the predominantly occurring descriptor, with the KRFPC314 fingerprint following closely in prevalence within the drug profiles of the CRC cell lines. Beyond predictive modeling, we also confirmed the applicability of our developed QSAR models via methods by conducting descriptor-drug analyses and recapitulating drug-to-oncogene relationships. We also identified two potential anti-CRC FDA-approved drugs, viomycin and diamorphine, using QSAR models. To ensure the easy accessibility and utility of our research findings, we have incorporated these models into a user-friendly prediction Web server named "ColoRecPred", available at https://project.iith.ac.in/cgntlab/colorecpred. We anticipate that this Web server can be used for screening of chemical libraries to identify potential anti-CRC drugs.
PubMed: 38680332
DOI: 10.1021/acsomega.4c01195