Relocalizing transcriptional kinases to activate apoptosis.

Kinases are critical regulators of cellular function that are commonly implicated in the mechanisms underlying disease. Most drugs that target kinases are molecules that inhibit their catalytic activity, but here we used chemically induced proximity to convert kinase inhibitors into activators of therapeutic genes. We synthesized bivalent molecules that link ligands of the transcription factor B cell lymphoma 6 (BCL6) to inhibitors of cyclin-dependent kinases (CDKs). These molecules relocalized CDK9 to BCL6-bound DNA and directed phosphorylation of RNA polymerase II. The resulting expression of pro-apoptotic, BCL6-target genes caused killing of diffuse large B cell lymphoma cells and specific ablation of the BCL6-regulated germinal center response. Genomics and proteomics corroborated a gain-of-function mechanism in which global kinase activity was not inhibited but rather redirected. Thus, kinase inhibitors can be used to context-specifically activate transcription.

Exploration of the tunability of BRD4 degradation by DCAF16 trans-labelling covalent glues.

Chemically induced proximity modalities such as targeted protein degradation (TPD) hold promise for expanding the number of proteins that can be manipulated pharmacologically. However, current TPD strategies are often limited to proteins with preexisting ligands. Molecular glues (e.g. glutarimide ligands for CUL4CRBN), offer the potential to target undruggable proteins. Yet, their rational design is largely unattainable due to the unpredictability of the 'gain-of-function' nature of the glue interaction upon chemical modification of ligands. We recently reported a covalent trans-labelling glue mechanism which we named 'Template-assisted covalent modification', where an electrophile decorated BRD4 inhibitor was effectively delivered to a cysteine residue on DCAF16 due to an electrophile-induced BRD4-DCAF16 interaction. Herein, we report our efforts to evaluate how various electrophilic modifications to the BRD4 binder, JQ1, affect DCAF16 recruitment and subsequent BRD4 degradation efficiency. We discovered a moderate correlation between the electrophile-induced BRD4-DCAF16 ternary complex formation and BRD4 degradation. Moreover, we show that a more solvent-exposed warhead presentation optimally recruits DCAF16 and promotes BRD4 degradation. The diversity of covalent attachments in this class of BRD4 degraders suggests a high tolerance and tunability for the BRD4-DCAF16 interaction. This offers a new avenue for rational glue design by introducing covalent warheads to known binders.

Unveiling the hidden interactome of CRBN molecular glues with chemoproteomics.

Targeted protein degradation and induced proximity refer to strategies that leverage the recruitment of proteins to facilitate their modification, regulation or degradation. As prospective design of glues remains challenging, unbiased discovery methods are needed to unveil hidden chemical targets. Here we establish a high throughput affinity purification mass spectrometry workflow in cell lysates for the unbiased identification of molecular glue targets. By mapping the targets of 20 CRBN-binding molecular glues, we identify 298 protein targets and demonstrate the utility of enrichment methods for identifying novel targets overlooked using established methods. We use a computational workflow to estimate target confidence and perform a biochemical screen to identify a lead compound for the new non-ZF target PPIL4. Our study provides a comprehensive inventory of targets chemically recruited to CRBN and delivers a robust and scalable workflow for identifying new drug-induced protein interactions in cell lysates.

DCLK1 induces a pro-tumorigenic phenotype to drive gastric cancer progression.

Doublecortin-like kinase 1 (DCLK1) is a proposed driver of gastric cancer (GC) that phosphorylates serine and threonine residues. Here, we showed that the kinase activity of DCLK1 orchestrated cancer cell-intrinsic and-extrinsic processes that led to pro-invasive and pro-metastatic reprogramming of GC cells. Inhibition of the kinase activity of DCLK1 reduced the growth of subcutaneous xenograft tumors formed from MKN1 human gastric carcinoma cells in mice and decreased the abundance of the stromal markers α-Sma, vimentin, and collagen. Similar effects were seen in mice with xenograft tumors formed from MKN1 cells expressing a kinase-inactive DCLK1 mutant (MKN1D511N). MKN1D511N cells also had reduced in vitro migratory potential and stemness compared with control cells. Mice orthotopically grafted with MKN1 cells overexpressing DCLK1 (MKN1DCLK1) showed increased invasiveness and had a greater incidence of lung metastases compared with those grafted with control MKN1 cells. Mechanistically, we showed that the chemokine CXCL12 acted downstream of DCLK1 in cultured MKN1 cells and in mice subcutaneously implanted with gastric tumors formed by MKN1DCLK1 cells. Moreover, inhibition of the kinase activity of DCLK1 or the expression of DCLK1D511N reversed the pro-tumorigenic and pro-metastatic phenotype. Together, this study establishes DCLK1 as a broadly acting and potentially targetable promoter of GC.

Discovery of Potent Degraders of the Dengue Virus Envelope Protein.

Targeted protein degradation has been widely adopted as a new approach to eliminate both established and previously recalcitrant therapeutic targets. Here, it is reported that the development of small molecule degraders of the envelope (E) protein of dengue virus. Two classes of bivalent E-degraders are developed by linking two previously reported E-binding small molecules, GNF-2, and CVM-2-12-2, to a glutarimide-based recruiter of the CRL4CRBN ligase to effect proteosome-mediated degradation of the E protein. ZXH-2-107 (based on GNF-2) is an E-degrader with ABL inhibitory activity while ZXH-8-004 (based on CVM-2-12-2) is a selective and potent E-degrader. These two compounds provide proof of concept that difficult-to-drug targets such as a viral envelope protein can be effectively eliminated using a bivalent degrader and provide starting points for the future development of a new class of direct-acting antiviral drugs.

Molecular Bidents with Two Electrophilic Warheads as a New Pharmacological Modality.

A systematic strategy to develop dual-warhead inhibitors is introduced to circumvent the limitations of conventional covalent inhibitors such as vulnerability to mutations of the corresponding nucleophilic residue. Currently, all FDA-approved covalent small molecules feature one electrophile, leaving open a facile route to acquired resistance. We conducted a systematic analysis of human proteins in the protein data bank to reveal ∼400 unique targets amendable to dual covalent inhibitors, which we term "molecular bidents". We demonstrated this strategy by targeting two kinases: MKK7 and EGFR. The designed compounds, ZNL-8162 and ZNL-0056, are ATP-competitive inhibitors that form two covalent bonds with cysteines and retain potency against single cysteine mutants. Therefore, molecular bidents represent a new pharmacological modality with the potential for improved selectivity, potency, and drug resistance profile.

Template-assisted covalent modification underlies activity of covalent molecular glues.

Molecular glues are proximity-inducing small molecules that have emerged as an attractive therapeutic approach. However, developing molecular glues remains challenging, requiring innovative mechanistic strategies to stabilize neoprotein interfaces and expedite discovery. Here we unveil a trans-labeling covalent molecular glue mechanism, termed 'template-assisted covalent modification'. We identified a new series of BRD4 molecular glue degraders that recruit CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). Through comprehensive biochemical, structural and mutagenesis analyses, we elucidated how pre-existing structural complementarity between DCAF16 and BRD4BD2 serves as a template to optimally orient the degrader for covalent modification of DCAF16Cys58. This process stabilizes the formation of BRD4-degrader-DCAF16 ternary complex and facilitates BRD4 degradation. Supporting generalizability, we found that a subset of degraders also induces GAK-BRD4BD2 interaction through trans-labeling of GAK. Together, our work establishes 'template-assisted covalent modification' as a mechanism for covalent molecular glues, which opens a new path to proximity-driven pharmacology.

Discovery of bivalent small molecule degraders of cyclin-dependent kinase 7 (CDK7).

Cyclin-dependent kinase 7, along with cyclin H and MAT1, forms the CDK-activating complex (CAK), which directs cell cycle progression via T-loop phosphorylation of cell cycle CDKs. Pharmacological inhibition of CDK7 leads to selective anti-cancer effects in cellular and in vivo models, motivating several ongoing clinical investigations of this target. Current CDK7 inhibitors are either reversible or covalent inhibitors of its catalytic activity. We hypothesized that small molecule targeted protein degradation (TPD) might result in differentiated pharmacology due to the loss of scaffolding functions. Here, we report the design and characterization of a potent CDK7 degrader that is comprised of an ATP-competitive CDK7 binder linked to a CRL2VHL recruiter. JWZ-5-13 effectively degrades CDK7 in multiple cancer cells and leads to a potent inhibition of cell proliferation. Additionally, compound JWZ-5-13 displayed bioavailability in a pharmacokinetic study conducted in mice. Therefore, JWZ-5-13 is a useful chemical probe to investigate the pharmacological consequences of CDK7 degradation.

Discovery of Potent Degraders of the Dengue Virus Envelope Protein.

Targeted protein degradation has been widely adopted as a new approach to eliminate both established and previously recalcitrant therapeutic targets. Here we report the development of small molecule degraders of the envelope (E) protein of dengue virus. We developed two classes of bivalent E-degraders, linking two previously reported E-binding small molecules, GNF-2 and CVM-2-12-2, to a glutarimide-based recruiter of the CRL4CRBN ligase to effect proteosome-mediated degradation of the E protein. ZXH-2-107 (based on GNF-2) is an E degrader with ABL inhibition while ZXH-8-004 (based on CVM-2-12-2) is a selective and potent E-degrader. These two compounds provide proof-of-concept that difficult-to-drug targets such as a viral envelope protein can be effectively eliminated using a bivalent degrader and provide starting points for the future development of a new class antiviral drugs.

Broad-spectrum activity against mosquito-borne flaviviruses achieved by a targeted protein degradation mechanism.

Viral genetic diversity presents significant challenges in developing antivirals with broad-spectrum activity and high barriers to resistance. Here we report development of proteolysis targeting chimeras (PROTACs) targeting the dengue virus envelope (E) protein through coupling of known E fusion inhibitors to ligands of the CRL4CRBN E3 ubiquitin ligase. The resulting small molecules block viral entry through inhibition of E-mediated membrane fusion and interfere with viral particle production by depleting intracellular E in infected Huh 7.5 cells. This activity is retained in the presence of point mutations previously shown to confer partial resistance to the parental inhibitors due to decreased inhibitor-binding. The E PROTACs also exhibit broadened spectrum of activity compared to the parental E inhibitors against a panel of mosquito-borne flaviviruses. These findings encourage further exploration of targeted protein degradation as a differentiated and potentially advantageous modality for development of broad-spectrum direct-acting antivirals.

Down-Regulation of AKT Proteins Slows the Growth of Mutant-KRAS Pancreatic Tumors.

Serine/threonine kinase AKT isoforms play a well-established role in cell metabolism and growth. Most pancreatic adenocarcinomas (PDACs) harbor activation mutations of KRAS, which activates the PI3K/AKT signaling pathway. However, AKT inhibitors are not effective in the treatment of pancreatic cancer. To better understand the role of AKT signaling in mutant-KRAS pancreatic tumors, this study utilized proteolysis-targeting chimeras (PROTACs) and CRISPR-Cas9-genome editing to investigate AKT proteins. The PROTAC down-regulation of AKT proteins markedly slowed the growth of three pancreatic tumor cell lines harboring mutant KRAS. In contrast, the inhibition of AKT kinase activity alone had very little effect on the growth of these cell lines. The concurrent genetic deletion of all AKT isoforms (AKT1, AKT2, and AKT3) in the KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cell line also dramatically slowed its growth in vitro and when orthotopically implanted in syngeneic mice. Surprisingly, insulin-like growth factor-1 (IGF-1), but not epidermal growth factor (EGF), restored KPC cell growth in serum-deprived conditions, and the IGF-1 growth stimulation effect was AKT-dependent. The RNA-seq analysis of AKT1/2/3-deficient KPC cells suggested that reduced cholesterol synthesis may be responsible for the decreased response to IGF-1 stimulation. These results indicate that the presence of all three AKT isoforms supports pancreatic tumor cell growth, and the pharmacological degradation of AKT proteins may be more effective than AKT catalytic inhibitors for treating pancreatic cancer.

Down-regulation of AKT proteins slows the growth of mutant-KRAS pancreatic tumors.

Serine/threonine kinase AKT isoforms play a well-established role in cell metabolism and growth. Most pancreatic adenocarcinoma (PDAC) harbors activation mutations of KRAS, which activates the PI3K/AKT signaling pathway. However, AKT inhibitors are not effective in the treatment of pancreatic cancer. To better understand the role of AKT signaling in mutant-KRAS pancreatic tumors, this study utilizes proteolysis-targeting chimeras (PROTACs) and CRISPR-Cas9-genome editing to investigate AKT proteins. PROTAC down-regulation of AKT proteins markedly slowed the growth of three pancreatic tumor cell lines harboring mutant KRAS. In contrast, inhibition of AKT kinase activity alone had very little effect on the growth of these cell lines. Concurrent genetic deletion of all AKT isoforms (AKT1, AKT2, and AKT3) in the KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cell line also dramatically slowed its growth in vitro and when orthotopically implanted in syngeneic mice. Surprisingly, insulin-like growth factor-1 (IGF-1), but not epidermal growth factor (EGF), restored KPC cell growth in serum-deprived conditions and the IGF-1 growth stimulation effect was AKT dependent. RNA-seq analysis of AKT1/2/3-deficient KPC cells suggested that reduced cholesterol synthesis may be responsible for the decreased response to IGF-1 stimulation. These results indicate that the presence of all three AKT isoforms supports pancreatic tumor cell growth and pharmacological degradation of AKT proteins may be more effective than AKT catalytic inhibitors for treating pancreatic cancer.

Proteasomal control of anti-CRISPRs for the regulation of CRISPR/Cas9 activity using Cas9-ACROBAT.

Small molecule-mediated proteasomal degradation of proteins is a powerful tool for synthetic regulation of biological activity. To control Cas9 activity in cells, we engineered an anti-CRISPR protein, AcrIIA4, fused to a degradation (dTAG) or small molecule assisted shutoff (SMASh) tag. Co-expression of the tagged AcrIIA4 along with Cas9 and riboswitch-regulated sgRNAs enables precise tunable control of CRISPR activity by small molecule addition.

Discovery of CRBN-dependent WEE1 Molecular Glue Degraders from a Multicomponent Combinatorial Library.

Small molecules promoting protein-protein interactions produce a range of therapeutic outcomes. Molecular glue degraders exemplify this concept due to their compact drug-like structures and ability to engage targets without reliance on existing cognate ligands. While Cereblon molecular glue degraders containing glutarimide scaffolds have been approved for treatment of multiple myeloma and acute myeloid leukemia, the design of new therapeutically relevant monovalent degraders remains challenging. We report here an approach to glutarimide-containing molecular glue synthesis using multicomponent reactions as a central modular core-forming step. Screening the resulting library identified HRZ-01 derivatives that target casein kinase 1 alpha (CK1α) and Wee-like protein kinase (WEE1). Further medicinal chemistry efforts led to identification of selective monovalent WEE1 degraders that provide a potential starting point for the eventual development of a selective chemical degrader probe. The structure of the hit WEE1 degrader complex with CRBN-DDB1 and WEE1 provides a model of the protein-protein interface and a rationale for the observed kinase selectivity. Our findings suggest that modular synthetic routes combined with in-depth structural characterization give access to selective molecular glue degraders and expansion of the CRBN-degradable proteome.

Reciprocal antagonism of PIN1-APC/CCDH1 governs mitotic protein stability and cell cycle entry.

Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/CCDH1) prevents cell-cycle entry by targeting crucial mitotic proteins for degradation. Phosphorylation of its co-activator CDH1 modulates the E3 ligase activity, but little is known about its regulation after phosphorylation and how to effectively harness APC/CCDH1 activity to treat cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1)-catalyzed phosphorylation-dependent cis-trans prolyl isomerization drives tumor malignancy. However, the mechanisms controlling its protein turnover remain elusive. Through proteomic screens and structural characterizations, we identify a reciprocal antagonism of PIN1-APC/CCDH1 mediated by domain-oriented phosphorylation-dependent dual interactions as a fundamental mechanism governing mitotic protein stability and cell-cycle entry. Remarkably, combined PIN1 and cyclin-dependent protein kinases (CDKs) inhibition creates a positive feedback loop of PIN1 inhibition and APC/CCDH1 activation to irreversibly degrade PIN1 and other crucial mitotic proteins, which force permanent cell-cycle exit and trigger anti-tumor immunity, translating into synergistic efficacy against triple-negative breast cancer.

The dual HCK/BTK inhibitor KIN-8194 impairs growth and integrin-mediated adhesion of BTKi-resistant mantle cell lymphoma.

Although Bruton's tyrosine kinase (BTK) inhibitors (BTKi) have significantly improved patient prognosis, mantle cell lymphoma (MCL) is still considered incurable due to primary and acquired resistance. We have recently shown that aberrant expression of the Src-family tyrosine kinase hematopoietic cell kinase (HCK) in MCL correlates with poor prognosis, and that genetic HCK perturbation impairs growth and integrin-mediated adhesion of MCL cells. Here, we show that KIN-8194, a dual inhibitor of BTK and HCK with in vivo activity against Myd88-L265P-driven diffuse large B-cell lymphoma and Waldenström Macroglobulinemia, has a potent growth inhibitory effect in MCL cell lines and primary MCL cells, irrespective of their sensitivity to BTKi (ibrutinib and acalabrutinib). In BTKi-resistant cells this is mediated by inhibition of HCK, which results in repression of AKT-S6 signaling. In addition, KIN-8194 inhibits integrin-mediated adhesion of BTKi-sensitive and insensitive MCL cells to fibronectin and stromal cells in an HCK-dependent manner. Finally, we show that MCL cells with acquired BTKi resistance retain their sensitivity to KIN-8194. Taken together, our data demonstrate that KIN-8194 inhibits growth and integrin-mediated adhesion of BTKi-sensitive MCL cells, as well as MCL cells with primary or acquired BTKi resistance. This renders KIN-8194 a promising novel treatment for MCL patients.

Targeting DCAF5 suppresses SMARCB1-mutant cancer by stabilizing SWI/SNF.

Whereas oncogenes can potentially be inhibited with small molecules, the loss of tumour suppressors is more common and is problematic because the tumour-suppressor proteins are no longer present to be targeted. Notable examples include SMARCB1-mutant cancers, which are highly lethal malignancies driven by the inactivation of a subunit of SWI/SNF (also known as BAF) chromatin-remodelling complexes. Here, to generate mechanistic insights into the consequences of SMARCB1 mutation and to identify vulnerabilities, we contributed 14 SMARCB1-mutant cell lines to a near genome-wide CRISPR screen as part of the Cancer Dependency Map Project1-3. We report that the little-studied gene DDB1-CUL4-associated factor 5 (DCAF5) is required for the survival of SMARCB1-mutant cancers. We show that DCAF5 has a quality-control function for SWI/SNF complexes and promotes the degradation of incompletely assembled SWI/SNF complexes in the absence of SMARCB1. After depletion of DCAF5, SMARCB1-deficient SWI/SNF complexes reaccumulate, bind to target loci and restore SWI/SNF-mediated gene expression to levels that are sufficient to reverse the cancer state, including in vivo. Consequently, cancer results not from the loss of SMARCB1 function per se, but rather from DCAF5-mediated degradation of SWI/SNF complexes. These data indicate that therapeutic targeting of ubiquitin-mediated quality-control factors may effectively reverse the malignant state of some cancers driven by disruption of tumour suppressor complexes.

ZNL0325, a Pyrazolopyrimidine-Based Covalent Probe, Demonstrates an Alternative Binding Mode for Kinases.

The pyrazolopyrimidine (PP) heterocycle is a versatile and widely deployed core scaffold for the development of kinase inhibitors. Typically, a 4-amino-substituted pyrazolopyrimidine binds in the ATP-binding pocket in a conformation analogous to the 6-aminopurine of ATP. Here, we report the discovery of ZNL0325 which exhibits a flipped binding mode where the C3 position is oriented toward the ribose binding pocket. ZNL0325 and its analogues feature an acrylamide side chain at the C3 position which is capable of forming a covalent bond with multiple kinases that possess a cysteine at the αD-1 position including BTK, EGFR, BLK, and JAK3. These findings suggest that the ability to form a covalent bond can override the preferred noncovalent binding conformation of the heterocyclic core and provides an opportunity to create structurally distinct covalent kinase inhibitors.

Multiomic profiling of breast cancer cells uncovers stress MAPK-associated sensitivity to AKT degradation.

More than 50% of human tumors display hyperactivation of the serine/threonine kinase AKT. Despite evidence of clinical efficacy, the therapeutic window of the current generation of AKT inhibitors could be improved. Here, we report the development of a second-generation AKT degrader, INY-05-040, which outperformed catalytic AKT inhibition with respect to cellular suppression of AKT-dependent phenotypes in breast cancer cell lines. A growth inhibition screen with 288 cancer cell lines confirmed that INY-05-040 had a substantially higher potency than our first-generation AKT degrader (INY-03-041), with both compounds outperforming catalytic AKT inhibition by GDC-0068. Using multiomic profiling and causal network integration in breast cancer cells, we demonstrated that the enhanced efficacy of INY-05-040 was associated with sustained suppression of AKT signaling, which was followed by induction of the stress mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK). Further integration of growth inhibition assays with publicly available transcriptomic, proteomic, and reverse phase protein array (RPPA) measurements established low basal JNK signaling as a biomarker for breast cancer sensitivity to AKT degradation. Together, our study presents a framework for mapping the network-wide signaling effects of therapeutically relevant compounds and identifies INY-05-040 as a potent pharmacological suppressor of AKT signaling.