Type II Binders Targeting the “GLR-Out” Conformation of the Pseudokinase STRADα

Abstract

Pseudokinases play important roles in signal transduction and cellular processes similar to those of catalytically competent kinases. However, pseudokinase pharmacological tractability and conformational space accessibility are poorly understood. Pseudokinases have only recently been suggested to adopt “inactive” conformations or interact with conformation-specific kinase inhibitors (e.g., type II compounds). In this work, the heavily substituted pseudokinase STRADα, which possesses a DFG → GLR substitution in the catalytic site that permits nucleotide binding while impairing divalent cation coordination, is used as a test case to demonstrate the potential applicability of conformation-specific, type II compounds to pseudokinase pharmacology. Integrated structural modeling is employed to generate a “GLR-out” conformational ensemble. Likely interacting type II compounds are identified through virtual screening against this ensemble model. Biophysical validation of compound binding is demonstrated through protein thermal stabilization and ATP competition. Localization of a top-performing compound through surface methylation strongly suggests that STRADα can adopt the “GLR-out” conformation and interact with compounds that comply with the standard type II pharmacophore. These results suggest that, despite a loss of catalytic function, some pseudokinases, including STRADα, may retain the conformational switching properties of conventional protein kinases.

There’s more to death than life: Noncatalytic functions in kinase and pseudokinase signaling

“…For example, compounds that occlude MLKL pseudokinase binding by RIPK3 kinase—emulating the function of viral MLKL—are plausible necroptosis pathway inhibitors, but may prove challenging to identify. One prospect for discovery of such inhibitors is virtual screening, as recently illustrated in the identification and validation of STRADα pseudoactive site binders ( 108 ). Alternatively fragment screening may be appropriate, which has been widely used for conventional kinases but, to our knowledge, not yet reported for pseudokinase studies.…”

Design and Development of a Chemical Probe for Pseudokinase Ca2+/calmodulin-Dependent Ser/Thr Kinase

“…Thus, the potential role of CASK in cancer may be addressed perturbing its scaffolding function by, for instance, developing a GFG-out type-II inhibitor as recently demonstrated for the pseudokinase STRAD which also harbors an unusual DFG motif (GLR). 42 The presence of an additional glycine in CASK may facilitate the GFG-out movement due to the increase flexibility by this glycine-rich sequence. A potential target of allosteric inhibitors would be the association of the CASK/Mint1/Munc18-1 ternary complex, a protein complex that involves the CASK pseudokinase domain which is induced by glucose stimulation in pancreatic β cells regulating insulin secretion.…”

“…Interestingly, in differentiated cells, or in cancer cells, compound 26 showed no toxicity, even at higher concentrations, suggesting that CASK catalytic activity was not required for cell survival. Thus, the potential role of CASK in cancer may be addressed perturbing its scaffolding function by, for instance, developing a GFG-out type-II inhibitor as recently demonstrated for the pseudokinase STRAD which also harbors an unusual DFG motif (GLR) . The presence of an additional glycine in CASK may facilitate the GFG-out movement due to the increase flexibility by this glycine-rich sequence.…”

Gaussian field-based 3D-QSAR and molecular simulation studies to design potent pyrimidine–sulfonamide hybrids as selective BRAFV600Einhibitors

“…Dabrafenib inhibits BRAF V600E most potently but is less effective against BRAF V600WT . 40,41 In 2011, vemurafenib, a pyrolo-pyrimidine based diphenyl sulfonamide derivative 42 of type I 1/2 , (aC-OUT/ DFG-IN) 43 received FDA approval for rst oral BRAF V600E inhibitor. 44,45 Encorafenib (LGX-818), a pyrazolo-pyrimidine based phenyl sulfonamide derivative, 39 was approved by the FDA in June 2018 for the treatment of BRAF V600E/K mutated cancer.…”