Targeted protein degradation (TPD) refers to the use of small molecules to induce ubiquitin-dependent degradation of proteins. TPD is of interest in drug development, as it can address previously ...inaccessible targets. However, degrader discovery and optimization remains an inefficient process due to a lack of understanding of the relative importance of the key molecular events required to induce target degradation. Here, we use chemo-proteomics to annotate the degradable kinome. Our expansive dataset provides chemical leads for ∼200 kinases and demonstrates that the current practice of starting from the highest potency binder is an ineffective method for discovering active compounds. We develop multitargeted degraders to answer fundamental questions about the ubiquitin proteasome system, uncovering that kinase degradation is p97 dependent. This work will not only fuel kinase degrader discovery, but also provides a blueprint for evaluating targeted degradation across entire gene families to accelerate understanding of TPD beyond the kinome.
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•A global map of kinase degradability provides chemical leads for >200 kinases•Open-access chemical proteomics resource (https://proteomics.fischerlab.org)•Large-scale chemical exploration of key variables for targeted protein degradation•Multi-targeted degraders uncover fundamentals of ubiquitin-mediated protein turnover
A synthetic chemistry and chemo-proteomics platform used to annotate the “degradable kinome” provides chemical leads for developing degraders of approximately 200 distinct kinase targets and offers new general design principles for developing future kinase degraders.
Pancreatic ductal adenocarcinoma (PDAC) is characterized by notorious resistance to current therapies attributed to inherent tumor heterogeneity and highly desmoplastic and immunosuppressive tumor ...microenvironment (TME). Unique proline isomerase Pin1 regulates multiple cancer pathways, but its role in the TME and cancer immunotherapy is unknown. Here, we find that Pin1 is overexpressed both in cancer cells and cancer-associated fibroblasts (CAFs) and correlates with poor survival in PDAC patients. Targeting Pin1 using clinically available drugs induces complete elimination or sustained remissions of aggressive PDAC by synergizing with anti-PD-1 and gemcitabine in diverse model systems. Mechanistically, Pin1 drives the desmoplastic and immunosuppressive TME by acting on CAFs and induces lysosomal degradation of the PD-1 ligand PD-L1 and the gemcitabine transporter ENT1 in cancer cells, besides activating multiple cancer pathways. Thus, Pin1 inhibition simultaneously blocks multiple cancer pathways, disrupts the desmoplastic and immunosuppressive TME, and upregulates PD-L1 and ENT1, rendering PDAC eradicable by immunochemotherapy.
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•Pin1 is overexpressed both in PDAC cells and CAFs and correlates with poor patient survival•Targeting Pin1 disrupts the desmoplastic and immunosuppressive TME by acting on CAFs•Targeting Pin1 increases PD-L1 and ENT1 expression in cancer cells by acting on HIP1R•Targeting Pin1 renders aggressive PDAC eradicable by synergizing with immunochemotherapy
Pin1 can drive the desmoplastic and immunosuppressive tumor microenvironment in addition to its known oncogenic functions.
Phosphatidylinositol 5‐phosphate 4‐kinase, type II, gamma (PIP4K2C) remains a poorly understood lipid kinase with minimal enzymatic activity but potential scaffolding roles in immune modulation and ...autophagy‐dependent catabolism. Achieving potent and selective agents for PIP4K2C while sparing other lipid and non‐lipid kinases has been challenging. Here, we report the discovery of the highly potent PIP4K2C binder TMX‐4102, which shows exclusive binding selectivity for PIP4K2C. Furthermore, we elaborated the PIP4K2C binder into TMX‐4153, a bivalent degrader capable of rapidly and selectively degrading endogenous PIP4K2C. Collectively, our work demonstrates that PIP4K2C is a tractable and degradable target, and that TMX‐4102 and TMX‐4153 are useful leads to further interrogate the biological roles and therapeutic potential of PIP4K2C.
One obstacle to a better understanding of the “dark” lipid kinase PIP4K2C is the lack of chemical tool compounds for this protein. Here, we describe a picomolar PIP4K2C binder TMX‐4102, and an effective bivalent PIP4K2C degrader, TMX‐4153, both of which display exclusive binding selectivity for PIP4K2C. Thus, TMX‐4102 and TMX‐4153 represent two useful leads to further interrogate the therapeutic potential of PIP4K2C.
The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. ...Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.
Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in ...mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity and cell permeability to give BJP-06-005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.
The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of ...potent, specific small molecules available. Here, we present the discovery and characterization of a pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4Kα/β/γ. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K's reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders.
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•Inhibitor THZ-P1-2 shows PI5P4K enzyme inhibition and target engagement in cells•THZ-P1-2 covalently targets unannotated cysteines outside the PI5P4K active site•AML/ALL cell lines are broadly sensitive to THZ-P1-2's covalent effects•PI5P4K inhibition causes autophagy disruption and upregulates TFEB signaling
PI5P4K, an understudied kinase family, is essential in various disease contexts. Sivakumaren et al. develop and characterize PI5P4K inhibitor THZ-P1-2, which targets unique cysteines, exhibits effects in biochemical and cellular assays, displays anticancer activity in leukemia cell lines, and causes defects in autophagy similar to PI5P4K gene knockdown or deletion.
Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks), a family of three members in mammals (α, β and γ), have emerged as potential therapeutic targets due to their role in regulating many important ...cellular signaling pathways. In comparison to the PI5P4Kα and PI5P4Kβ, which usually have similar expression profiles across cancer cells, PI5P4Kγ exhibits distinct expression patterns, and pathological functions for PI5P4Kγ have been proposed in the context of cancer and neurodegenerative diseases. PI5P4Kγ has very low kinase activity and has been proposed to inhibit the PI4P5Ks through scaffolding function, providing a rationale for developing a selective PI5P4Kγ degrader. Here, we report the development and characterization of JWZ-1-80, a first-in-class PI5P4Kγ degrader. JWZ-1-80 potently degrades PI5P4Kγ via the ubiquitin-proteasome system and exhibits proteome-wide selectivity and is therefore a useful tool compound for further dissecting the biological functions of PI5P4Kγ.
JWZ-1-80 induces rapid and sustained proteasomal degradation of endogenous PI5P4Kγ through recruitment of the E3 ligase adaptor VHL. JWZ-1-80 exhibits excellent proteome-wide selectivity and does not degrade PI5P4Kα and PI5P4Kβ. Display omitted
•A first-in-class PI5P4Kγ degrader were developed.•JWZ-1-80 selectively degraded PI5P4Kγwithout affecting PI5P4Kα and PI5P4Kβ.•JWZ-1-80 degraded PI5P4Kγ through the ubiquitin-protease pathway.•JWZ-1-80 exhibits high proteome-wide selectivity.•JWZ-1-80 induced the degradation of PI5P4Kγ in multiple cell lines.
Due to their role in many important signaling pathways, phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are attractive targets for the development of experimental therapeutics for cancer, ...metabolic, and immunological disorders. Recent efforts to develop small molecule inhibitors for these lipid kinases resulted in compounds with low- to sub-micromolar potencies. Here, we report the identification of CVM-05-002 using a high-throughput screen of PI5P4Kα against our in-house kinase inhibitor library. CVM-05-002 is a potent and selective inhibitor of PI5P4Ks, and a 1.7 Å X-ray structure reveals its binding interactions in the ATP-binding pocket. Further investigation of the structure–activity relationship led to the development of compound 13, replacing the rhodanine-like moiety present in CVM-05-002 with an indole, a potent pan-PI5P4K inhibitor with excellent kinome-wide selectivity. Finally, we employed isothermal cellular thermal shift assays (CETSAs) to demonstrate the effective cellular target engagement of PI5P4Kα and -β by the inhibitors in HEK 293T cells.
Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are important molecular players in a variety of diseases, such as cancer. Currently available PI5P4K inhibitors are reversible small molecules, ...which may lack selectivity and sufficient cellular on-target activity. In this study, we present a new class of covalent pan-PI5P4K inhibitors with potent biochemical and cellular activity. Our designs are based on THZ-P1-2, a covalent PI5P4K inhibitor previously developed in our lab. Here, we report further structure-guided optimization and structure–activity relationship (SAR) study of this scaffold, resulting in compound 30, which retained biochemical and cellular potency, while demonstrating a significantly improved selectivity profile. Furthermore, we confirm that the inhibitors show efficient binding affinity in the context of HEK 293T cells using isothermal CETSA methods. Taken together, compound 30 represents a highly selective pan-PI5P4K covalent lead molecule.
Phosphatidylinositol 5‐phosphate 4‐kinase, type II, gamma (PIP4K2C) remains a poorly understood lipid kinase with minimal enzymatic activity but potential scaffolding roles in immune modulation and ...autophagy‐dependent catabolism. Achieving potent and selective agents for PIP4K2C while sparing other lipid and non‐lipid kinases has been challenging. Here, we report the discovery of the highly potent PIP4K2C binder TMX‐4102, which shows exclusive binding selectivity for PIP4K2C. Furthermore, we elaborated the PIP4K2C binder into TMX‐4153, a bivalent degrader capable of rapidly and selectively degrading endogenous PIP4K2C. Collectively, our work demonstrates that PIP4K2C is a tractable and degradable target, and that TMX‐4102 and TMX‐4153 are useful leads to further interrogate the biological roles and therapeutic potential of PIP4K2C.
One obstacle to a better understanding of the “dark” lipid kinase PIP4K2C is the lack of chemical tool compounds for this protein. Here, we describe a picomolar PIP4K2C binder TMX‐4102, and an effective bivalent PIP4K2C degrader, TMX‐4153, both of which display exclusive binding selectivity for PIP4K2C. Thus, TMX‐4102 and TMX‐4153 represent two useful leads to further interrogate the therapeutic potential of PIP4K2C.
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