Parallel synthesis of indolylquinones and their cell-based insulin mimicry.

A synthetic route to bis-indolyldihydroxybenzoquinones was adapted for parallel organic synthesis. The route involves selective conjugate addition of an indole to dichlorobenzoquinone promoted by Brønsted acid, followed by a Lewis acid-promoted conjugate addition of a second indole and a final hydrolysis. Methods for high-throughput purification of the products of this synthesis were also developed. Using these methods, we prepared a library whose structures are based on asterriquinone natural products, which have a wide range of biological activities. In this report, the activities of the library members in activation of the insulin receptor on mammalian cells were examined. Novel compounds were discovered that fall outside earlier developed structure-activity relationships for insulin mimics, supporting the value of systematic investigation (inspired by Nature) for the discovery of novel biologically active molecules.

Alpha-crystallin binds to the aggregation-prone molten-globule state of alkaline protease: implications for preventing irreversible thermal denaturation.

Alpha-crystallin, the major eye-lens protein with sequence homology with heat-shock proteins (HSPs), acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain more insight into its chaperoning ability, we used a protease as the model system that is known to require a propeptide (intramolecular chaperone) for its proper folding. The protease ("N" state) from Conidiobolus macrosporus (NCIM 1298) unfolds at pH 2.0 ("U" state) through a partially unfolded "I" state at pH 3.5 that undergoes transition to a molten globule-(MG) like "I(A)" state in the presence of 0.5 M sodium sulfate. The thermally-stressed I(A) state showed complete loss of structure and was prone to aggregation. Alpha-crystallin was able to bind to this state and suppress its aggregation, thereby preventing irreversible denaturation of the enzyme. The alpha-crystallin-bound I(A) state exhibited native-like secondary and tertiary structure showing the interaction of alpha-crystallin with the MG state of the protease. 8-Anilinonaphthalene sulphonate (ANS) binding studies revealed the involvement of hydrophobic interactions in the formation of the complex of alpha-crystallin and protease. Refolding of acid-denatured protease by dilution to pH 7.5 resulted in aggregation of the protein. Unfolding of the protease in the presence of alpha-crystallin and its subsequent refolding resulted in the generation of a near-native intermediate with partial secondary and tertiary structure. Our studies represent the first report of involvement of a molecular chaperone-like alpha-crystallin in the unfolding and refolding of a protease. Alpha-crystallin blocks the unfavorable pathways that lead to irreversible denaturation of the alkaline protease and keeps it in a near-native, folding-competent intermediate state.