Cytochrome P450 (CYP) family of redox enzymes metabolize drugs and xenobiotics in liver microsomes. Isozyme CYP2C9 is reported to be inhibited by benzbromarone (BzBr) and this phenomenon was hitherto explained by classical active-site binding. Theoretically, it was impossible to envisage the experimentally derived sub-nM Ki for an inhibitor, when supra-nM enzyme and 10X KM substrate concentrations were employed. We set out to find a more plausible explanation for this highly intriguing “super-inhibition” phenomenon. In silico docking of various BzBr analogs with known crystal structure of CYP2C9 did not provide any evidence in support of active-site based inhibition hypothesis. Experiments tested the effects of BzBr and nine analogs on CYPs in reconstituted systems of lab-purified proteins, complex baculosomes & crude microsomal preparations. In certain setups, BzBr and its analogs could even enhance reactions, which cannot be explained by an active site hypothesis. Generally, it was seen that Ki became smaller by orders of magnitude, upon increasing the dilution order of BzBr analogs. Also, it was seen that BzBr could also inhibit other CYP isozymes like CYP3A4, CYP2D6 and CYP2E1. Further, amphipathic derivatives of vitamins C & E (scavengers of diffusible reactive oxygen species or DROS) effectively inhibited CYP2C9 reactions in different reaction setups. Therefore, the inhibition of CYP activity by BzBr analogs (which are also surface-active redox agents) is attributed to catalytic scavenging of DROS at phospholipid interface. The current work expands the scope of interpretations of inhibitions in redox enzymes and ushers in a new cellular biochemistry paradigm that small amounts of DROS may be obligatorily required in routine redox metabolism for constructive catalytic roles.

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Carboxylated nanoparticles play an important role in separation technology, catalysis, proton conduction, water purification, ion transport, and gas separation. Reported herein are small cyclic dipeptide templates appended with monomeric norbornene units for obtaining robust carboxylated nanospheres. The stereochemistry of the dipeptides dictates the size of the resulting nanoassembly. The exposed monomeric units on the nanospheres are polymerized to covalently capture the self-assembly. Removal of the peptide template via ester hydrolysis results in carboxylated polymeric spheres. Polymer cross-linking enhances the thermal stability of the spherical assembly, while the five-membered ring obtained after norbornene polymerization retains its shape even after removal of the template. The ability of these charged spheres to bind cations is illustrated by the binding of a positively charged chloride-sensing lucigenin dye. The chloride-sensing ability of the bound dye is not compromised, illustrating the utility of these nanospheres as potential chloride sensors. One can also envision extending this concept for binding and separating other charged species of interest.