coumermycin and coumarin

Development of heat shock protein 90 (Hsp90) C-terminal inhibitors has emerged as an exciting strategy for the treatment of cancer. Previous efforts have focused on modifications to the natural products novobiocin and coumermycin. Moreover, variations in both the sugar and amide moieties have been extensively studied, whereas replacements for the coumarin core have received less attention. Herein, 24 cores were synthesized with varying distances and angles between the sugar and amide moieties. Compounds that exhibited good anti-proliferative activity against multiple cancer cell lines and Hsp90 inhibitory activity, were those that placed the sugar and amide moieties between 7.7 and 12.1 Å apart along with angles of 180°.

Topics: Aminocoumarins; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Chemistry Techniques, Synthetic; Coumarins; Drug Screening Assays, Antitumor; HSP90 Heat-Shock Proteins; Humans; Molecular Structure; Novobiocin; Structure-Activity Relationship

A gene encoding the outer membrane lipoprotein, OmlA, from the bacterial phytopathogen Xanthomonas campestris pv. phaseoli was isolated and characterized. An omlA insertion mutant showed an increased susceptibility to novobiocin and coumermycin, antibiotics with gyrase inhibitor activity. The omlA mutant accumulated novobiocin. Additionally, the omlA mutant was more sensitive than the wild type to chloramphenicol, a protein synthesis inhibitor; SDS, a detergent; and menadione, a superoxide generator. The susceptibility of the mutant to unrelated chemicals indicated a general role for OmlA in maintaining membrane integrity. Transcription of omlA was downregulated in the presence of both gyrase inhibitors, suggesting that DNA supercoiling might regulate the synthesis of OmlA. The omlA gene was divergently transcribed from the gene encoding the ferric uptake regulator Fur. Although the promoters of omlA and fur overlapped, Fur did not play any regulatory role in the expression of omlA due to the fact that inactivation of Fur did not affect the expression of omlA either in the presence or absence of iron.

Topics: Aminocoumarins; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Base Sequence; Chloramphenicol; Coumarins; DNA, Bacterial; Drug Resistance, Multiple, Bacterial; Gene Expression; Lipoproteins; Microbial Sensitivity Tests; Molecular Sequence Data; Mutagenesis, Insertional; Novobiocin; Promoter Regions, Genetic; Sodium Dodecyl Sulfate; Transcription Initiation Site; Transcription, Genetic; Vitamin K 3; Xanthomonas campestris

The C-terminal domain of Hsp90 displays independent chaperone activity, mediates dimerization, and contains the MEEVD motif essential for interaction with tetratricopeptide repeat-containing immunophilin cochaperones assembled in mature steroid receptor complexes. An alpha-helical region, upstream of the MEEVD peptide, helps form the dimerization interface and includes a hydrophobic microdomain that contributes to the Hsp90 interaction with the immunophilin cochaperones and corresponds to the binding site for novobiocin, a coumarin-related Hsp90 inhibitor. Mutation of selected residues within the hydrophobic microdomain significantly impacted the chaperone function of a recombinant C-terminal Hsp90 fragment and novobiocin inhibited wild-type chaperone activity. Prior incubation of the Hsp90 fragment with novobiocin led to a direct blockade of immunophilin cochaperone binding. However, the drug had little influence on the pre-formed Hsp90-immunophilin complex, suggesting that bound cochaperones mask the novobiocin-binding site. We observed a differential effect of the drug on Hsp90-immunophilin interaction, suggesting that the immunophilins make distinct contacts within the C-terminal domain to specifically modulate Hsp90 function. Novobiocin also precluded the interaction of full-length Hsp90 with the p50(cdc37) cochaperone, which targets the N-terminal nucleotide-binding domain, and is prevalent in Hsp90 complexes with protein kinase substrates. Novobiocin therefore acts locally and allosterically to induce conformational changes within multiple regions of the Hsp90 protein. We provide evidence that coumermycin A1, a coumarin structurally related to novobiocin, interferes with dimerization of the Hsp90 C-terminal domain. Coumarin-based inhibitors then may antagonize Hsp90 function by inducing a conformation favoring separation of the C-terminal domains and release of substrate.

Topics: Alanine; Amino Acid Sequence; Aminocoumarins; Anti-Bacterial Agents; Binding Sites; Coumarins; Dimerization; DNA, Complementary; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Glutathione Transferase; HeLa Cells; HSP90 Heat-Shock Proteins; Humans; Immunophilins; Molecular Chaperones; Molecular Conformation; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Novobiocin; Plasmids; Point Mutation; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Steroids; Substrate Specificity; Tacrolimus Binding Proteins; Time Factors