PIFtide specifically binds to phosphoinositide-dependent kinase 1 (PDK1)

PIFtide is a synthetic peptide derived from the “hydrophobic motif region” or “peptide docking motif” of the protein kinase PKN2 (or PRK2), which specifically binds to phosphoinositide-dependent kinase 1 (PDK1). PIFtide with sequence REPRILSEEEQEMFRDFDYIADWC binds to the PIF pocket of the protein kinase PDK1.

Crystal structure of PDK1 in complex with ATP and PIFtide.

Complex of the polypeptide PIFtide (in green, derived from the HM of the protein kinase C-related protein kinase PRK2) with the PIF pocket of PDK1 (Rettenmaier et al.; PDB 4RRV).

The PIF pocket is a substrate recognition site in the kinase domain of PDK1. PKN2 is part of the protein kinase superfamily composed of the catalytic domains of serine/threonine-specific and tyrosine-specific protein kinases. PIFtide and phosphoinositide-dependent kinase 1 (PDK1) interaction indicates a signaling step. The serine/threonine kinase PDK1 is pivotal in various signal transduction pathways. Phosphatidylinositides often activate PDK1.

The PDK1 signaling pathway is a downstream effector of the PI3K signaling pathway, including various cellular functions such as cell cycle progression, metabolism, and autophagy. PDK1 is activated when a conserved serine residue in its activation loop is phosphorylated. PDK1 is translocated to the plasma membrane when phosphoinositide 3-kinase (PI3K) produces phosphatidylinositol-3,4,5-trisphosphate (PIP3). PDK1 phosphorylates other AGC protein kinases as well, such as protein kinase B (PKB)/Akt, p70 and p90 ribosomal S6 kinases (S6K1 and RSK), serum-and glucocorticoid-induced protein kinase (SGK), and protein kinase C (PKC) isoforms. The PDK1 signaling pathway is closely associated with many human cancers. PDK1 is essential for embryogenesis and life in adulthood in mammals. Also, PDK1 plays a role in regulating cardiac hyperpolarization-activated cyclic nucleotide-modulated channels.

Targeting allosteric sites may allow the design of selective inhibitors involving protein-protein or protein-peptide interfaces.

Rettenmaier et al. (2014) discovered molecules that structurally mimic the natural peptide ligand and inhibit PDK1 in cells. The research group observed that combining allosteric and ATP-competitive inhibitors blocked the activation of oncogenic kinases downstream of PDK1. Rettenmaier et al. presented a systematic approach for targeting a functionally conserved allosteric site on the protein kinase PDK1 called the PDK1-interacting fragment (PIF)tide-binding site, or PIF pocket by solving the first high-resolution structure of a peptide docking motif (PIFtide) bound to PDK1 and mapped binding energy hot spots using mutational analysis.

The structures of PDK1 bound to allosteric small molecules revealed a binding mode that mimics three of five hot-spot residues in PIFtide. These allosteric small molecules are substrate-selective PDK1 inhibitors when used as single agents, but when combined with an ATP-competitive inhibitor, they suppress entirely the activation of the downstream kinases.

To develop a suitable PIFtide probe for a fluorescent peptide binding assay, Rettenmaier et al. synthesized a panel of fluorescent PIFtides of various lengths and amino acid content. They found that the removal of the first 13 amino acids, the substitution of Phe14 and Phe17 with bromo-Phe residues, the substitution of Tyr19 with Trp, and the conjugation of 6-tetramethylrhodamine to the N terminus resulted in a PIFtide probe with an ideal affinity and dynamic range (Kd = 40 nM; ΔmP = 130 mP). Tetramethylrhodamine, a red-shifted fluorophore relative to fluorescein, reduces the susceptibility of the assay to interference from autofluorescent compounds. The resulting competitive binding assay allowed a robust high-throughput screen.

                         Polypeptide Substrates for PDK1

Rettenmaier et al. suggested that this approach could be adapted to target any analogous allosteric site found on many other kinases. 

Allosteric Regulation of PDK1

The allosteric regulation of phosphoinositide-dependent protein kinase 1 (PDK1) involves small molecules and macromolecules, such as peptides binding to specific sites on the protein. These interactions cause changes in other parts of the protein, even though they're not in direct contact.

The allosteric regulation of PDK1 involves the PIF pocket, a conserved allosteric site on PDK1 that regulates its activity, ATP binding, and interactions with substrates.

Also, small molecules can bind to the ATP-binding site and affect the PIF pocket. Macromolecular partners, such as regulatory proteins, can bind to PDK1 and cause allosteric changes. Further, regulatory motifs in the kinase-PH interdomain linker can activate PDK1 autophosphorylation, and  PIP3 binding drives switch-like activation of PDK1. 

PDK1 is a kinase that phosphorylates and activates other kinases. These kinases regulate cell proliferation, metabolism, and survival. PDK1 is an important target for anticancer drugs. Allosteric modulators of PDK1 are RS1 and RS2, ligands stimulating PDK1 activity toward a short peptide substrate, 2-O-benzyl-IP5, an inositol phosphate-based potent inhibitor of PDK1, and 2-O-benzoyl-IP5 (HYG8), a synthetic IP5 inhibitor of PDK1. 

References

4RRV

Mingione VR, Paung Y, Outhwaite IR, Seeliger MA. Allosteric regulation and inhibition of protein kinases. Biochem Soc Trans. 2023 Feb 27;51(1):373-385. [PMC]

Rettenmaier TJ, Sadowsky JD, Thomsen ND, Chen SC, Doak AK, Arkin MR, Wells JA. A small-molecule mimic of a peptide docking motif inhibits the protein kinase PDK1. Proc Natl Acad Sci U S A. 2014 Dec 30;111(52):18590-5. [PubMed]

Turek-Etienne TC, Small EC, Soh SC, Xin TA, Gaitonde PV, Barrabee EB, Hart RF, Bryant RW. Evaluation of fluorescent compound interference in 4 fluorescence polarization assays: 2 kinases, 1 protease, and 1 phosphatase. J Biomol Screen. 2003 Apr;8(2):176-84. [PubMed]

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