A Protoberberine Derivative HWY336 Selectively Inhibits MKK4 and MKK7 in Mammalian Cells: The Importance of Activation Loop on Selectivity

Abstract

A protoberberine derivative library was used to search for selective inhibitors against kinases of the mitogen-activated protein kinase (MAPK) cascades in mammalian cells. Among kinases in mammalian MAPK pathways, we identified a compound (HWY336) that selectively inhibits kinase activity of mitogen-activated protein kinase kinase 4 and 7 (MKK4 and MKK7). The IC50 of HWY336 was 6 µM for MKK4 and 10 µM for MKK7 in vitro. HWY336 bound to both kinases reversibly via noncovalent interactions, and inhibited their activity by interfering with access of a protein substrate to its binding site. The binding affinity of HWY336 to MKK4 was measured by surface plasmon resonance to determine a dissociation constant (Kd) of 3.2 µM. When mammalian cells were treated with HWY336, MKK4 and MKK7 were selectively inhibited, resulting in inhibition of c-Jun NH2-terminal protein kinases in vivo. The structural model of HWY336 bound to either MKK4 or MKK7 predicted that HWY336 was docked to the activation loop, which is adjacent to the substrate binding site. This model suggested the importance of the activation loop of MKKs in HWY336 selectivity. We verified this model by mutating three critical residues within this loop of MKK4 to the corresponding residues in MKK3. The mutant MKK4 displayed similar kinase activity as wild-type kinase, but its activity was not inhibited by HWY336 compared to wild-type MKK4. We propose that the specific association of HWY336 to the activation loop of MKK4/MKK7 is responsible for its selective inhibition.

Direct interaction of Ste11 and Mkk1/2 through Nst1 integrates high‐osmolarity glycerol and pheromone pathways to the cell wall integrity MAPK pathway

“…Ste11‐GFP and Mkk1‐HA were immune‐precipitated as described in coimmunoprecipitation assays. The kinase reaction was performed by mixing 5 μL of the purified kinase Ste11‐GFP and 20 μL of the purified substrate Mkk1‐HA or mutants in a kinase reaction buffer as described . After 30 min at 30 °C, the reaction was stopped by adding SDS sample buffer and boiling.…”

Synthesis and Biological Evaluation of 3‐Arylindazoles as Selective MEK4 Inhibitors

“…To date, in the literature there has been minimal advancement in MEK4 inhibitor development. HWY336 ( 1 ), a protoberberine derivative, inhibits both MEK4 and MEK7 (Figure ) . HWY336 not only has poor selectivity and only moderate potency, but the pharmacological parameters are not ideal as it is a tetracyclic alkaloid, a compounds class known for promiscuity in biological effects .…”

“…HWY336 (1), ap rotoberberine derivative, inhibits both MEK4 and MEK7 ( Figure 1). [8] HWY336 not only has poor selectivity and only moderate potency,b ut the pharmacological parameters are not ideal as it is at etracyclic alkaloid, ac ompounds class known for promiscuity in biological effects. [9] Trihydroxyisoflavones have also been shownt oh ave effects against MEK4 but not in as elective manner.…”

Emerging role of the Jun N‐terminal kinase interactome in human health

“…THIF may serve as a potential chemo‐preventive agent in skin cancer by suppressing the occurrence and multiplicity of UVB‐induced skin tumors in hairless mice (Lee et al, ). The protoberberine derivative HWY336 inhibits both MKK4 and MKK7, with high selectivity to wild‐type MKK4 (Kim et al, ). Vacquinols are synthesized quinine derivatives that activate MKK4 in glioblastoma cells to induce glioma cell death.…”