Family-wide Structural Analysis of Human Numb-Associated Protein Kinases

Abstract

The highly diverse Numb-associated kinase (NAK) family has been linked to broad cellular functions including receptor-mediated endocytosis, Notch pathway modulation, osteoblast differentiation, and dendrite morphogenesis. Consequently, NAK kinases play a key role in a diverse range of diseases from Parkinson's and prostate cancer to HIV. Due to the plasticity of this kinase family, NAK kinases are often inhibited by approved or investigational drugs and have been associated with side effects, but they are also potential drug targets. The presence of cysteine residues in some NAK family members provides the possibility for selective targeting via covalent inhibition. Here we report the first high-resolution structures of kinases AAK1 and BIKE in complex with two drug candidates. The presented data allow a comprehensive structural characterization of the NAK kinase family and provide the basis for rational design of selective NAK inhibitors.

Keywords: AAK1; BIKE; Numb-associated kinase (NAK); activation segment; inhibitor selectivity.

Graphical abstract

Figure 1

Sequence Conservation and Domain Organization of NAK Family (A) NAK family phylogenetic tree. C.e., Caenorhabditis elegans; D.m., Drosophila melanogaster; H.s., Homo sapiens; M.m., Mus musculus; S.c., Saccharomyces cerevisiae. (B) Domain organization of human NAKs. (C) Sequence alignment of human NAK catalytic domains colored by residue conservation. Secondary structure as well as structural elements important for NAK function and inhibitor interactions are highlighted (green arrow, β sheet; red cylinder, α helix; blue cylinders, NAK-specific helices; yellow triangle indicates the active-site cysteine important for covalent inhibitor binding; red hexagon indicates regulatory spine residues; residues boxed in orange indicate the position of mutated residues in BIKE that have been introduced to aid crystallization).

Figure 2

Crystal Structures of Human NAK Family Kinase Domains (A) Overlay of crystal structures of AAK1 (gray, PDB: 4wsq), BIKE (orange, PDB: 4w9w), GAK (green, PDB: 4o57), and MPSK1 (pink, PDB: 2buj) showing location of the activation segment C-terminal helix (ASCH). See also Figure S1. (B) Comparison of ASCH in MPSK1 versus AAK1. (C) Alignment of R spine in BIKE. (D) Sedimentation velocity of phosphorylated AAK1 showing that it is monomeric in solution. (E) Sequence and structure alignment of NAKs, with conserved residues shown in violet, non-conserved residues in white, non-aligned residues in gray.

Figure 3

Clinical Kinase Inhibitor Binding to NAKs (A) Thermal shift assay data for a selection of clinical inhibitors against each of the NAK family kinase domains. See also Table S1 and Figure S2. (B) ITC determination of thermodynamic parameters for inhibitor compounds determined at 15°C. (C) ITC data measured for the interaction of nintedanib with AAK1 and BIKE. The data showed a 10-fold difference in affinity. (D) The structure of nintedanib. (E) The structure of SP600125.

Figure 4

Small-Molecule Inhibitor Interactions with AAK1 and BIKE (A) Interaction of K252a with AAK1. Inhibitors are shown in stick representation with yellow carbon atoms. Hydrogen bonds are indicated by dotted lines. Key interacting residues are shown and labeled. (B) Chemical structures of K252a and related compound lestaurtinib. (C and D) Interaction of BIKE with (C) AZD7762 with (D) baricitinib. Inhibitors are shown in stick representation with yellow carbon atoms. Hydrogen bonds are indicated by dotted lines. Key interacting residues are shown and labeled. (E) Chemical structure of (5Z)-7-oxozeaenol. (F) Denaturing mass spectra for NAKs in the presence of (5Z)-7-oxozeaenol.