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Nucleic Acids Research Oct 2023Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a...
Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a first-in-class inhibitor of the Polθ ATPase activity, and it is currently being tested in clinical trials as an anti-cancer drug. Here, we investigated the molecular mechanism of NVB-mediated Polθ inhibition. Using hydrogen deuterium exchange-mass spectrometry (HX-MS), biophysical, biochemical, computational and cellular assays, we found NVB is a non-competitive inhibitor of ATP hydrolysis. NVB sugar group deletion resulted in decreased potency and reduced HX-MS interactions, supporting a specific NVB binding orientation. Collective results revealed that NVB binds to an allosteric site to block DNA binding, both in vitro and in cells. Comparisons of The Cancer Genome Atlas (TCGA) tumors and matched controls implied that POLQ upregulation in tumors stems from its role in replication stress responses to increased cell proliferation: this can now be tested in fifteen tumor types by NVB blocking ssDNA-stimulation of ATPase activity, required for Polθ function at replication forks and DNA damage sites. Structural and functional insights provided in this study suggest a path for developing NVB derivatives with improved potency for Polθ inhibition by targeting ssDNA binding with entropically constrained small molecules.
Topics: Humans; Adenosine Triphosphatases; DNA Polymerase theta; DNA Replication; DNA, Single-Stranded; DNA-Directed DNA Polymerase; Neoplasms; Novobiocin
PubMed: 37665033
DOI: 10.1093/nar/gkad727 -
Virology Sep 2020Chikungunya has re-emerged as an epidemic with global distribution and high morbidity, necessitating the need for effective therapeutics. We utilized already approved...
Chikungunya has re-emerged as an epidemic with global distribution and high morbidity, necessitating the need for effective therapeutics. We utilized already approved drugs with a good safety profile used in other diseases for their new property of anti-chikungunya activity. It provides a base for a fast and efficient approach to bring a novel therapy from bench to bedside by the process of drug-repositioning. We utilized an in-silico drug screening with FDA approved molecule library to identify inhibitors of the chikungunya nsP2 protease, a multifunctional and essential non-structural protein required for virus replication. Telmisartan, an anti-hypertension drug, and the antibiotic novobiocin emerged among top hits on the screen. Further, SPR experiments revealed strong in-vitro binding of telmisartan and novobiocin to nsP2 protein. Additionally, small angle x-ray scattering suggested binding of molecules to nsP2 and post-binding compaction and retention of monomeric state in the protein-inhibitor complex. Protease activity measurement revealed that both compounds inhibited nsP2 protease activity with IC values in the low micromolar range. More importantly, plaque formation assays could show the effectiveness of these drugs in suppressing virus propagation in host cells. We propose novobiocin and telmisartan as potential inhibitors of chikungunya replication. Further research is required to establish the molecules as antivirals of clinical relevance against chikungunya.
Topics: Antiviral Agents; Chikungunya Fever; Chikungunya virus; Drug Evaluation, Preclinical; Humans; Novobiocin; Telmisartan; Viral Nonstructural Proteins; Virus Replication
PubMed: 32791353
DOI: 10.1016/j.virol.2020.05.010 -
International Journal of Antimicrobial... Jul 2020Burkholderia pseudomallei causes melioidosis, a potentially lethal disease that can establish both chronic and acute infections in humans. It is inherently recalcitrant...
Burkholderia pseudomallei causes melioidosis, a potentially lethal disease that can establish both chronic and acute infections in humans. It is inherently recalcitrant to many antibiotics, there is a paucity of effective treatment options and there is no vaccine. In the present study, the efficacies of selected aminocoumarin compounds, DNA gyrase inhibitors that were discovered in the 1950s but are not in clinical use for the treatment of melioidosis were investigated. Clorobiocin and coumermycin were shown to be particularly effective in treating B. pseudomallei infection in vivo. A novel formulation with dl-tryptophan or l-tyrosine was shown to further enhance aminocoumarin potency in vivo. It was demonstrated that coumermycin has superior pharmacokinetic properties compared with novobiocin, and the coumermycin in l-tyrosine formulation can be used as an effective treatment for acute respiratory melioidosis in a murine model. Repurposing of existing approved antibiotics offers new resources in a challenging era of drug development and antimicrobial resistance.
Topics: Aminocoumarins; Animals; Burkholderia pseudomallei; Disease Models, Animal; Drug Resistance, Multiple, Bacterial; Drug Therapy, Combination; Female; Melioidosis; Mice; Mice, Inbred BALB C; Moths; Novobiocin; Tryptophan
PubMed: 32361027
DOI: 10.1016/j.ijantimicag.2020.106002 -
Scientific Reports Jul 2018Fibronectin (FN) plays a major role in the stability and organization of the extracellular matrix (ECM). We have previously demonstrated that FN interacts directly with...
Fibronectin (FN) plays a major role in the stability and organization of the extracellular matrix (ECM). We have previously demonstrated that FN interacts directly with Hsp90, as well as showing that the Hsp90 inhibitor novobiocin results in FN turnover via a receptor mediated process. However, the receptor involved has not been previously identified. LRP1 is a ubiquitous receptor responsible for the internalisation of numerous ligands that binds both Hsp90 and FN, and therefore we investigated whether LRP1 was involved in novobiocin-mediated FN turnover. FN, LRP1 and Hsp90 could be isolated in a common complex, and inhibition of Hsp90 by novobiocin increased the colocalisation of FN and LRP1. Novobiocin induced an increase (at low concentrations) followed by a loss of FN that was primarily derived from extracellular matrix-associated FN and led to a concomitant increase in intracellular FN. The effect of novobiocin was specific to LRP1-expressing cells and could be recapitulated by an LRP1 blocking antibody and the allosteric C-terminal Hsp90 inhibitor SM253, but not the N-terminal inhibitor geldanamycin. Together these data suggest that LRP1 is required for FN turnover in response to Hsp90 inhibition by novobiocin, which may have unintended physiological consequences in contexts where C-terminal Hsp90 inhibition is to be used therapeutically.
Topics: Animals; Antibodies, Blocking; Cell Line; Endocytosis; Extracellular Matrix; Extracellular Space; Fibronectins; HSP90 Heat-Shock Proteins; Humans; Low Density Lipoprotein Receptor-Related Protein-1; Mice; Novobiocin; Proteolysis
PubMed: 30061663
DOI: 10.1038/s41598-018-29531-2 -
The Journal of Antimicrobial... Oct 2020To evaluate the efficacy of two novel compounds against mycobacteria and determine the molecular basis of their action on DNA gyrase using structural and mechanistic...
OBJECTIVES
To evaluate the efficacy of two novel compounds against mycobacteria and determine the molecular basis of their action on DNA gyrase using structural and mechanistic approaches.
METHODS
Redx03863 and Redx04739 were tested in antibacterial assays, and also against their target, DNA gyrase, using DNA supercoiling and ATPase assays. X-ray crystallography was used to determine the structure of the gyrase B protein ATPase sub-domain from Mycobacterium smegmatis complexed with the aminocoumarin drug novobiocin, and structures of the same domain from Mycobacterium thermoresistibile complexed with novobiocin, and also with Redx03863.
RESULTS
Both compounds, Redx03863 and Redx04739, were active against selected Gram-positive and Gram-negative species, with Redx03863 being the more potent, and Redx04739 showing selectivity against M. smegmatis. Both compounds were potent inhibitors of the supercoiling and ATPase reactions of DNA gyrase, but did not appreciably affect the ATP-independent relaxation reaction. The structure of Redx03863 bound to the gyrase B protein ATPase sub-domain from M. thermoresistibile shows that it binds at a site adjacent to the ATP- and novobiocin-binding sites. We found that most of the mutations that we made in the Redx03863-binding pocket, based on the structure, rendered gyrase inactive.
CONCLUSIONS
Redx03863 and Redx04739 inhibit gyrase by preventing the binding of ATP. The fact that the Redx03863-binding pocket is distinct from that of novobiocin, coupled with the lack of activity of resistant mutants, suggests that such compounds could have potential to be further exploited as antibiotics.
Topics: Adenosine Triphosphatases; DNA Gyrase; Mycobacteriaceae; Mycobacterium; Novobiocin; Topoisomerase II Inhibitors
PubMed: 32728686
DOI: 10.1093/jac/dkaa286 -
Journal of Medicinal Chemistry Jan 2019Molecular chaperones HSP90 and HSP70 are essential regulators of the folding and activation of a disparate ensemble of client proteins. They function through ATP... (Review)
Review
Molecular chaperones HSP90 and HSP70 are essential regulators of the folding and activation of a disparate ensemble of client proteins. They function through ATP hydrolysis and the assembly of multiprotein complexes with cochaperones and clients. While their therapeutic relevance is recognized, important details underlying the links between ATP-dependent conformational dynamics and clients/cochaperones recruitment remain elusive. Allosteric modulators represent fundamental tools to obtain molecular insights into functional regulation. By selective perturbation of different aspects of HSP90/HSP70 activities, allosteric drugs can tune rather than completely inhibit signaling cascades, providing information on the relationships between structure-dynamics and function. Herein, we review advances in the design of HSP90 and HSP70 allosteric modulators. We consider inhibitors and activators in different biochemical and disease models. We discuss these compounds as probes to decipher the complexity of the chaperone machinery and that at the same time represent starting leads for the development of drugs against cancer and neurodegeneration.
Topics: Allosteric Regulation; Allosteric Site; Drug Design; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Molecular Dynamics Simulation; Novobiocin; Protein Structure, Tertiary; Pyridinium Compounds; Thiazoles
PubMed: 30048133
DOI: 10.1021/acs.jmedchem.8b00825 -
Proceedings of the National Academy of... Jun 2018Combinatorial drug treatment strategies perturb biological networks synergistically to achieve therapeutic effects and represent major opportunities to develop advanced...
Combinatorial drug treatment strategies perturb biological networks synergistically to achieve therapeutic effects and represent major opportunities to develop advanced treatments across a variety of human disease areas. However, the discovery of new combinatorial treatments is challenged by the sheer scale of combinatorial chemical space. Here, we report a high-throughput system for nanoliter-scale phenotypic screening that formulates a chemical library in nanoliter droplet emulsions and automates the construction of chemical combinations en masse using parallel droplet processing. We applied this system to predict synergy between more than 4,000 investigational and approved drugs and a panel of 10 antibiotics against , a model gram-negative pathogen. We found a range of drugs not previously indicated for infectious disease that synergize with antibiotics. Our validated hits include drugs that synergize with the antibiotics vancomycin, erythromycin, and novobiocin, which are used against gram-positive bacteria but are not effective by themselves to resolve gram-negative infections.
Topics: Anti-Bacterial Agents; Combinatorial Chemistry Techniques; Drug Discovery; Drug Evaluation, Preclinical; Drug Synergism; Erythromycin; Escherichia coli; High-Throughput Screening Assays; Lab-On-A-Chip Devices; Microarray Analysis; Microbial Sensitivity Tests; Nanotechnology; Novobiocin; Pseudomonas aeruginosa; Small Molecule Libraries; Vancomycin
PubMed: 29899149
DOI: 10.1073/pnas.1802233115 -
Antimicrobial Agents and Chemotherapy Jul 1986Because of the potential of novobiocin-rifampin for oral therapy of methicillin-resistant Staphylococcus aureus infection, we evaluated the pharmacokinetics of... (Clinical Trial)
Clinical Trial Randomized Controlled Trial
Because of the potential of novobiocin-rifampin for oral therapy of methicillin-resistant Staphylococcus aureus infection, we evaluated the pharmacokinetics of novobiocin and rifampin, alone and in combination, in a randomized, crossover, multiple-dose evaluation (500 mg of novobiocin and 300 mg of rifampin administered orally, twice a day, for 27 doses) in 10 volunteers. The half-lives of novobiocin and rifampin when administered alone were 5.85 +/- 1.20 and 1.46 +/- 0.30 h, respectively; when administered in combination, the half-lives were 2.66 +/- 0.65 and 1.43 +/- 0.29 h, respectively. This difference was significant for novobiocin. The area under the curve also differed significantly for novobiocin when administered in combination. No significant differences were seen in the maximum concentration of drug in serum, the time to maximum concentration of drug in serum, or both for either drug when single and combination therapy groups were compared. A change in clearance of novobiocin rather than a change in absorption is the more likely explanation for these findings. The mechanism remains to be elucidated. Nevertheless, the trough serum concentrations of both novobiocin and rifampin were in excess of the MIC for 90% of strains tested of methicillin-resistant S. aureus, even when coadministered.
Topics: Adolescent; Adult; Double-Blind Method; Drug Therapy, Combination; Humans; Kinetics; Male; Middle Aged; Novobiocin; Random Allocation; Rifampin
PubMed: 3752982
DOI: 10.1128/AAC.30.1.42 -
British Medical Journal Jul 1968
Topics: Drug Resistance, Microbial; Endocarditis, Bacterial; Fusidic Acid; Humans; Novobiocin; Staphylococcal Infections; Vancomycin
PubMed: 5661914
DOI: No ID Found -
Current Medicinal Chemistry 2008The 90 kDa heat shock proteins (Hsp90), which are integrally involved in cell signaling, proliferation, and survival, are ubiquitously expressed in cells. Many proteins... (Review)
Review
The 90 kDa heat shock proteins (Hsp90), which are integrally involved in cell signaling, proliferation, and survival, are ubiquitously expressed in cells. Many proteins in tumor cells are dependent upon the Hsp90 protein folding machinery for their stability, refolding, and maturation. Inhibition of Hsp90 uniquely targets client proteins associated with all six hallmarks of cancer. Thus, Hsp90 has emerged as a promising target for the treatment of cancer. Hsp90 exists as a homodimer, which contains three domains. The N-terminal domain contains an ATP-binding site that binds the natural products geldanamycin and radicicol. The middle domain is highly charged and has high affinity for co-chaperones and client proteins. Initial studies by Csermely and co-workers suggested a second ATP-binding site in the C-terminus of Hsp90. This C-terminal nucleotide binding pocket has been shown to not only bind ATP, but cisplatin, novobiocin, epilgallocatechin-3-gallate (EGCG) and taxol. The coumarin antibiotics novobiocin, clorobiocin, and coumermycin A1 were isolated from several streptomyces strains and exhibit potent activity against Gram-positive bacteria. These compounds bind type II topoisomerases, including DNA gyrase, and inhibit the enzyme-catalyzed hydrolysis of ATP. As a result, novobiocin analogues have garnered the attention of numerous researchers as an attractive agent for the treatment of bacterial infection. Novobiocin was reported to bind weakly to the newly discovered Hsp90 C-terminal ATP binding site ( approximately 700 M in SkBr3 cells) and induce degradation of Hsp90 client proteins. Structural modification of this compound has led to an increase of 1000-fold in activity in anti-proliferative assays. Recent studies of structure-activity relationship (SAR) by Renoir and co-workers highlighted the crucial role of the C-4 and/or C-7 positions of the coumarin and removal of the noviose moiety, which appeared to be essential for degradation of Hsp90 client proteins. Unlike the N-terminal ATP binding site, there is no reported co-crystal structure of Hsp90 C-terminus bound to any inhibitor. The Hsp90 C-terminal domain, however, is known to contain a conserved pentapeptide sequence (MEEVD) which is recognized by co-chaperones. Cisplatin is a platinum-containing chemotherapeutic used to treat various types of cancers, including testicular, ovarian, bladder, and small cell lung cancer. Most notably, cisplatin coordinates to DNA bases, resulting in cross-linked DNA, which prohibits rapidly dividing cells from duplicating DNA for mitosis. Itoh and co-workers reported that cisplatin decreases the chaperone activity of Hsp90. This group applied bovine brain cytosol to a cisplatin affinity column, eluted with cisplatin and detected Hsp90 in the eluent. Subsequent experiments indicated that cisplatin exhibits high affinity for Hsp90. Moreover Csermely and co-workers determined that the cisplatin binding site is located proximal to the C-terminal ATP binding site. EGCG is one of the active ingredients found in green tea. EGCG is known to inhibit the activity of many Hsp90-dependent client proteins, including telomerase, several kinases, and the aryl hydrocarbon receptor (AhR). Recently Gasiewicz and co-workers reported that EGCG manifests its antagonistic activity against AhR through binding Hsp90. Similar to novobiocin, EGCG was shown to bind the C-terminus of Hsp90. Unlike previously identified N-terminal Hsp90 inhibitors, EGCG does not appear to prevent Hsp90 from forming multiprotein complexes. Studies are currently underway to determine whether EGCG competes with novobiocin or cisplatin binding. Taxol, a well-known drug for the treatment of cancer, is responsible for the stabilization of microtubules and the inhibition of mitosis. Previous studies have shown that taxol induces the activation of kinases and transcription factors, and mimics the effect of bacterial lipopolysaccharide (LPS), an attribute unrelated to its tubulin-binding properties. Rosen and co-workers prepared a biotinylated taxol derivative and performed affinity chromatography experiments with lysates from both mouse brain and macrophage cell lines. These studies led to identification of two chaperones, Hsp70 and Hsp90, by mass spectrometry. In contrast to typical Hsp90-binding drugs, taxol exhibits a stimulatory response. Recently it was reported that the geldanamycin derivative 17-AAG behaves synergistically with taxol-induced apoptosis. This review describes the different C-terminal inhibitors of Hsp90, with specific emphasis on structure-activity relationship studies of novobiocin and their effects on anti-proliferative activity.
Topics: Adenosine Triphosphate; Animals; Binding Sites; HSP90 Heat-Shock Proteins; Humans; Neoplasms; Neurodegenerative Diseases; Novobiocin; Protein Binding
PubMed: 18991631
DOI: 10.2174/092986708786242895