Structure-Activity Studies of Novel di-substituted [1,2,5]oxadiazolo[3,4-b]pyrazine Analogs Targeting the A-loop Regulatory Site of p38 MAP Kinase

Introduction: In the quest for novel allosteric inhibitors of the p38 MAP kinase, we recently described the A-loop regulatory site, identified through molecular modeling studies together with the disclosure of a small molecule hit with a moderate inhibitory profile. Starting from this structure, we subsequently identified two additional hits with simpler molecular structures from an in silico screening study, using a substructure search in the SciFinder database. After corroboration of their inhibitory profile, analysis of their structures permitted to conclude about the suitability of the [1,2,5]oxadiazolo[3,4-b]pyrazine (furazano[3,4-b]pyrazine) scaffold for the development of potent A-loop regulatory site p38 MAP kinase inhibitors. Accordingly, we report the synthesis and pharmacological evaluation of a series of di-substituted analogs with a potent inhibitory profile of p38 MAP kinase, as shown by in vitro assays of their capability to inhibit IL-1β secretion in human monocyte-derived macrophages. Background: In the quest for novel allosteric inhibitors of the p38 MAP kinase, we recently described the A-loop regulatory site, identified through molecular modeling studies together with the disclosure of a small molecule hit with a moderate inhibitory profile. Objective: To find small molecule potent inhibitors of the p38 MAP kinase A-loop regulatory site. Methods: Starting from this structure, we subsequently identified two additional hits with simpler molecular structures from an in silico screening study, using a substructure search in the SciFinder database. After corroboration of their inhibitory profile, we carried out a hit-to-lead optimization process guided by molecular modeling using a [1,2,5]oxadiazolo[3,4-b]pyrazine (furazano[3,4-b]pyrazine) scaffold. Results: We report the synthesis and pharmacological evaluation of a series of di-substituted analogs with a potent inhibitory profile of p38 MAP kinase, as shown by in vitro assays of their capability to inhibit IL-1β secretion in human monocyte-derived macrophages. Conclusions: We describe in the present work a series of [1,2,5]oxadiazolo[3,4-b]pyrazine (furazano[3,4-b]pyrazine), potent inhibitors of IL-1β secretion in human monocyte-derived macrophages allosteric modulators of the p38 MAP kinase A-loop regulatory site.

Transcriptional co-activator regulates melanocyte differentiation and oncogenesis by integrating cAMP and MAPK/ERK pathways

SummaryThe cyclic AMP pathway promotes melanocyte differentiation in part by triggering gene expression changes mediated by CREB and its coactivators (CRTC1-3). Differentiation is dysregulated in melanomas, although the contributions of different cAMP effectors in this setting is unclear. We report a selective differentiation impairment in CRTC3 KO melanocytes and melanoma cells, due to downregulation of OCA2 and block of melanosome maturation. CRTC3 stimulated OCA2 expression via binding to CREB on a conserved enhancer, a regulatory site for pigmentation and melanoma risk in humans. Response to cellular signaling differed between CRTC3 and its family members; CRTC3 was uniquely activated by ERK1/2-mediated phosphorylation at Ser391 and by low levels of cAMP. Phosphorylation at Ser391 was constitutively elevated in human melanoma cells with hyperactivated ERK1/2 signaling; knockout of CRTC3 in this setting impaired anchorage-independent growth, migration and invasiveness while CRTC3 overexpression supported cell survival in response to MAPK inhibition by vemurafenib. Human melanomas expressing gain of function mutations in CRTC3 were associated with poorer clinical outcome. Our results suggest that CRTC3 inhibition may provide benefit in the treatment of hyperpigmentation and melanoma, and potentially other disorders with deregulated cAMP/MAPK crosstalk.