Diphencyprone Treatment of Alopecia Areata: Postulated Mechanism of Action and Prospects for Therapeutic Synergy with RNA Interference.

Diphencyprone (DPCP) is a potent topical sensitizing agent that has been used since the late 1970s by physicians for the treatment of alopecia areata (AA), viral warts (human papillomavirus) and cutaneous metastases of melanoma. Although to date the compound is not approved as a drug by the FDA or EMA, physicians have continued to use DPCP because of its proven effects in these dermatological conditions. The use of the drug has been highly variable because of differences in compounding, and as a result, the literature reports vary widely in the concentrations used for sensitization and challenge treatment with DPCP. The efficacy of DPCP has generally been ascribed to immunological reactions by the host. Inducing inflammation with a contact sensitizer is counterintuitive to treating AA, an autoimmune disorder. We have hypothesized that the body's attempt to downregulate the inflammation caused by the contact sensitizer may also ameliorate AA. Studies using microarray and miRNA profiling may provide information about how DPCP induces inflammation in human skin at different times. Gene targets and microRNAs identified through these data may be modulated by an RNA interference approach to enhance DPCP efficacy and response rates. In addition, this approach may result in the discovery and development of drugs that are more potent and selective for the treatment of AA.

The kinetic mechanism of the human bifunctional enzyme ATIC (5-amino-4-imidazolecarboxamide ribonucleotide transformylase/inosine 5'-monophosphate cyclohydrolase). A surprising lack of substrate channeling.

5-Amino-4-imidazolecarboxamide ribonucleotide transformylase/IMP cyclohydrolase (ATIC) is a bifunctional protein possessing two enzymatic activities that sequentially catalyze the last two steps in the pathway for de novo synthesis of inosine 5'-monophosphate. This bifunctional enzyme is of particular interest because of its potential as a chemotherapeutic target. Furthermore, these two catalytic activities reside on the same protein throughout all of nature, raising the question of whether there is some kinetic advantage to the bifunctionality. Rapid chemical quench, stopped-flow absorbance, and steady-state kinetic techniques were used to elucidate the complete kinetic mechanism of human ATIC. The kinetic simulation program KINSIM was used to model the kinetic data obtained in this study. The detailed kinetic analysis, in combination with kinetic simulations, provided the following key features of the enzyme reaction pathway. 1) The rate-limiting step in the overall reaction (2.9 +/- 0.4 s(-1)) is likely the release of tetrahydrofolate from the formyltransferase active site or a conformational change associated with tetrahydrofolate release. 2) The rate of the reverse transformylase reaction (6.7 s(-1)) is approximately 2-3-fold faster than the forward rate (2.9 s(-1)), whereas the cyclohydrolase reaction is essentially unidirectional in the forward sense. The cyclohydrolase reaction thus draws the overall bifunctional reaction toward the production of inosine monophosphate. 3) There was no kinetic evidence of substrate channeling of the intermediate, the formylaminoimidazole carboxamide ribonucleotide, between the formyltransferase and the cyclohydrolase active sites.