No abstract

Non-steroidal anti-inflammatory drugs and aureolic acid group of anti-cancer drugs belong to the class of generic drugs. Research with some members of these two groups of drugs in different laboratories has unveiled functions other than those for which they were primarily developed as drugs. Here we have reviewed the molecular mechanism behind the multiple functions of these drugs that might lead to employ them for treatment of diseases in addition to those they are presently employed.

SUMMARY The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme in complex with a σ factor that is required for promoter-specific transcription initiation. During initiation, members of the σ70 family of σ factors contact two conserved promoter elements, the −10 and −35 elements, that are separated by ~17 base pairs (bp). σ70 family members contain four flexibly linked domains. Two of these domains, σ2 and σ4, contain determinants for interactions with the promoter −10 and −35 elements, respectively. σ2 and σ4 also contain core-binding determinants. When bound to core the inter-domain distance between σ2 and σ4 matches the distance between promoter elements separated by ~17 bp. Prior work indicates that during early elongation the nascent RNA-assisted displacement of σ4 from core can enable the holoenzyme to adopt a configuration in which σ2 and σ4 are bound to “promoter-like” DNA elements separated by a single base pair. Here we demonstrate that holoenzyme can also adopt configurations in which σ2 and σ4 are bound to “promoter-like” DNA elements separated by 0, 2 or 3 bp. Thus, our findings suggest that displacement of σ4 from core enables the RNAP holoenzyme to adopt a broad range of “elongation-specific” configurations.

The aqueous-phase self-association of mithramycin (MTR), an aureolic acid anticancer antibiotic, has been studied using different spectroscopic techniques such as absorption, fluorescence, circular dichroism, and 1H nuclear magnetic resonance spectroscopy. Results from these studies indicate self-association of the anionic antibiotic at pH 8.0 over a concentration range from micromolar to millimolar. These results could be ascribed to the following steps of self-association: M + M left arrow over right arrow M2, M2 + M left arrow over right arrow M3, and M3 + M left arrow over right arrow M4, where M, M2, M3, and M4 represent the monomer, dimer, trimer, and tetramer of mithramycin, respectively. Dynamic light scattering and isothermal titration calorimetry studies also support aggregation. In contrast, an insignificant extent of self-association is found for the neutral drug (at pH 3.5) and the [(MTR)2Mg2+] complex (at pH 8.0). Analysis of 2D NMR spectra of 1 mM MTR suggests that the sugar moieties play a role in the self-association process. Self-association of the drug might occur either via hydrophobic interaction of the sugar residues among themselves or water-mediated hydrogen bond formation between sugar residue(s). On the other hand, absence of a significant upfield shift of the aromatic protons from 100 microM to 1 mM MTR suggests against the possibility of stacking interactions between the aromatic rings as a stabilizing force for the formation of the dimer and higher oligomers.

One of the major attributes for the biological action of the aureolic acid anticancer antibiotics chromomycin A(3) (CHR) and mithramycin (MTR) is their ability to bind bivalent cations such as Mg(II) and Zn(II) ions and form high affinity 2:1 complexes in terms of the antibiotic and the metal ion, respectively. As most of the cellular Zn(II) ion is found to be associated with proteins, we have examined the effect of MTR/CHR on the structure and function of a representative structurally well characterized Zn(II) metalloenzyme, alcohol dehydrogenase (ADH) from yeast. MTR and CHR inhibit enzyme activity of ADH with inhibitory constants of micromolar order. Results from size-exclusion column chromatography, dynamic light scattering, and isothermal titration calorimetry have suggested that the mechanism of inhibition of the metalloenzyme by the antibiotics is due to the antibiotic-induced disruption of the enzyme quaternary structure. The nature of the enzyme inhibition, the binding stoichiometry of two antibiotics per monomer, and comparable dissociation constants for the antibiotic and free (or substrate-bound) ADH imply that the association occurs as a consequence of the binding of the antibiotics to Zn(II) ion present at the structural center. Confocal microscopy shows the colocalization of the antibiotic and the metalloenzyme in HepG2 cells, thereby supporting the proposition of physical association between the antibiotic(s) and the enzyme inside the cell.