Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its potential as a therapeutic target. We leveraged chemoproteomic profiling and structure-based design to develop the first selective, in vivo -compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer.

We report that multi functional polymer nanoparticles approximately the size of a large protein can be "purified", on the basis of peptide affinity just as antibodies, using an affinity chromatography strategy. The selection process takes advantage of the thermo-responsiveness of the nanoparticles allowing "catch and release" of the target peptide by adjusting the temperature. Purified particles show much stronger affinity (Kd app ≈ nM) and a narrower affinity distribution than the average of particles before purification (Kd app > μM) in room temperature, but can release the peptide just by changing temperature. We anticipate this affinity selection will be general and become an integral step for the preparation of "plastic antibodies" with near homogeneous and tailored affinity for target biomacromolecules.General procedures for the creation of synthetic materials with biomacromolecular recognition sites are of significant interest as a route to stable, robust and mass-produced substitutes for antibodies. [1][2][3][4][5][6][7][8] Ideally, recognition of complex biological targets, including proteins, peptides and carbohydrates, requires multiple functional groups that contact target molecules by a combination of electrostatic, hydrogen-bonding, van der Waals, and/or hydrophobic interactions. It has been shown that copolymerization of optimized combinations and ratios of functional monomers creates synthetic polymer materials with molecular recognition sites. [1][2][3][4][5] However, in contrast to antibodies whose exact sequence can be determined and cloned, polymerized materials result in heterogeneous structures with a distribution of recognition sites. 1,3,7 This is an intrinsic property of polymers synthesized under kinetic control, in contrast to the synthetic small molecular hosts prepared by multi step reactions 9 or by self-assembly under equilibrating conditions 10 . Here we demonstrate a general procedure to purify synthetic polymer nanoparticles (NPs) with high-affinity binding sites for a target biomacromolecule from a random pool of multi-functional copolymer nanoparticles (MFNPs). These nanoparticles are approximately the size of a large protein and are "purified" on the basis of peptide affinity just as antibodies, using an affinity chromatography strategy. The concept of affinity purification of NPs was demonstrated with melittin, a 26 amino acid peptide ( fig. 1a), as the target molecule. Melittin has six positive charges of which four are localized in a hydrophilic six amino acid sequence on the C-terminus. The remaining twenty amino acids have a high proportion of apolar residues. 11For the MFNPs, we chose cross-linked N-isopropylacrylamide NPs (~30 nm) incorporating hydrophobic (N-t-butylacrylamide (TBAm)) and negatively charged (acrylic acid (AAc)) functional monomers ( fig. 1b). We have reported that NPs with this composition interacts with melittin (K dapp = 46 μM) via both electrostatic-and hydrophobi nteractions in PBS (35mM phosphate buffer/0.15 M NaCl, pH 7.3). 5 However, th...

Synthetic polymer nanoparticles with antibody-like affinity for a hydrophilic peptide have been prepared by inverse microemulsion polymerization. Peptide affinity was achieved in part by incorporating the target (imprint) peptide in the polymerization reaction mixture. Incorporation of the imprint peptide assists in the creation of complementary binding sites in the resulting polymer nanoparticle (NP). To orient the imprint peptide at the interface of the water and oil domains during polymerization, the peptide target was coupled with fatty acid chains of varying length. The peptide-NP binding affinities (90 nM~900 nM) were quantitatively evaluated by a quartz crystal microbalance (QCM). The optimal chain length was established that created high affinity peptide binding sites on the surface of the nanoparticles. This method can be used for the preparation of nanosized synthetic polymers with antibody-like affinity for hydrophilic peptides and proteins (“plastic antibodies”).