Discovery and characterization of a selective IKZF2 glue degrader for cancer immunotherapy.

Malignant tumors can evade destruction by the immune system by attracting immune-suppressive regulatory T cells (Treg) cells. The IKZF2 (Helios) transcription factor plays a crucial role in maintaining function and stability of Treg cells, and IKZF2 deficiency reduces tumor growth in mice. Here we report the discovery of NVP-DKY709, a selective molecular glue degrader of IKZF2 that spares IKZF1/3. We describe the recruitment-guided medicinal chemistry campaign leading to NVP-DKY709 that redirected the degradation selectivity of cereblon (CRBN) binders from IKZF1 toward IKZF2. Selectivity of NVP-DKY709 for IKZF2 was rationalized by analyzing the DDB1:CRBN:NVP-DKY709:IKZF2(ZF2 or ZF2-3) ternary complex X-ray structures. Exposure to NVP-DKY709 reduced the suppressive activity of human Treg cells and rescued cytokine production in exhausted T-effector cells. In vivo, treatment with NVP-DKY709 delayed tumor growth in mice with a humanized immune system and enhanced immunization responses in cynomolgus monkeys. NVP-DKY709 is being investigated in the clinic as an immune-enhancing agent for cancer immunotherapy.

Author Correction: CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Novel Luminescence-Based High-Throughput Approach for Cellular Resolution of Protein Ubiquitination Using Tandem Ubiquitin Binding Entities (TUBEs).

Protein turnover is highly regulated by the posttranslational process of ubiquitination. Deregulation of the ubiquitin proteasome system (UPS) has been implicated in cancer and neurodegenerative diseases, and modulating this system has proven to be a viable approach for therapeutic intervention. The development of novel technologies that enable high-throughput studies of substrate protein ubiquitination is key for UPS drug discovery. Conventional approaches for studying ubiquitination either have high protein requirements or rely on exogenous or modified ubiquitin moieties, thus limiting their utility. In order to circumvent these issues, we developed a high-throughput live-cell assay that combines the NanoBiT luminescence-based technology with tandem ubiquitin binding entities (TUBEs) to resolve substrate ubiquitination. To demonstrate the effectiveness and utility of this assay, we studied compound-induced ubiquitination of the G to S Phase Transition 1 (GSPT1) protein. Using this assay, we characterized compounds with varying levels of GSPT1 ubiquitination activity. This method provides a live-cell-based approach for assaying substrate ubiquitination that can be adapted to study the kinetics of ubiquitin transfer onto a substrate protein of interest. In addition, our results show that this approach is portable for studying the ubiquitination of target proteins with diverse functions.

Author Correction: Structural basis of indisulam-mediated RBM39 recruitment to DCAF15 E3 ligase complex.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structural basis of indisulam-mediated RBM39 recruitment to DCAF15 E3 ligase complex.

The anticancer agent indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor, leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which indisulam mediates the DCAF15-RBM39 interaction, we solved the DCAF15-DDB1-DDA1-indisulam-RBM39(RRM2) complex structure to a resolution of 2.3 Å. DCAF15 has a distinct topology that embraces the RBM39(RRM2) domain largely via non-polar interactions, and indisulam binds between DCAF15 and RBM39(RRM2), coordinating additional interactions between the two proteins. Studies with RBM39 point mutants and indisulam analogs validated the structural model and defined the RBM39 α-helical degron motif. The degron is found only in RBM23 and RBM39, and only these proteins were detectably downregulated in indisulam-treated HCT116 cells. This work further explains how indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade additional targets.

CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.

Discovery of Small Molecule Splicing Modulators of Survival Motor Neuron-2 (SMN2) for the Treatment of Spinal Muscular Atrophy (SMA).

Spinal muscular atrophy (SMA), a rare neuromuscular disorder, is the leading genetic cause of death in infants and toddlers. SMA is caused by the deletion or a loss of function mutation of the survival motor neuron 1 (SMN1) gene. In humans, a second closely related gene SMN2 exists; however it codes for a less stable SMN protein. In recent years, significant progress has been made toward disease modifying treatments for SMA by modulating SMN2 pre-mRNA splicing. Herein, we describe the discovery of LMI070/branaplam, a small molecule that stabilizes the interaction between the spliceosome and SMN2 pre-mRNA. Branaplam (1) originated from a high-throughput phenotypic screening hit, pyridazine 2, and evolved via multiparameter lead optimization. In a severe mouse SMA model, branaplam treatment increased full-length SMN RNA and protein levels, and extended survival. Currently, branaplam is in clinical studies for SMA.

Identification of NVP-TNKS656: the use of structure-efficiency relationships to generate a highly potent, selective, and orally active tankyrase inhibitor.

Tankyrase 1 and 2 have been shown to be redundant, druggable nodes in the Wnt pathway. As such, there has been intense interest in developing agents suitable for modulating the Wnt pathway in vivo by targeting this enzyme pair. By utilizing a combination of structure-based design and LipE-based structure efficiency relationships, the core of XAV939 was optimized into a more stable, more efficient, but less potent dihydropyran motif 7. This core was combined with elements of screening hits 2, 19, and 33 and resulted in highly potent, selective tankyrase inhibitors that are novel three pocket binders. NVP-TNKS656 (43) was identified as an orally active antagonist of Wnt pathway activity in the MMTV-Wnt1 mouse xenograft model. With an enthalpy-driven thermodynamic signature of binding, highly favorable physicochemical properties, and high lipophilic efficiency, NVP-TNKS656 is a novel tankyrase inhibitor that is well suited for further in vivo validation studies.

PIKfyve, a class III PI kinase, is the target of the small molecular IL-12/IL-23 inhibitor apilimod and a player in Toll-like receptor signaling.

Toll-like receptor (TLR) signaling is a key component of innate immunity. Aberrant TLR activation leads to immune disorders via dysregulation of cytokine production, such as IL-12/IL-23. Herein, we identify and characterize PIKfyve, a lipid kinase, as a critical player in TLR signaling using apilimod as an affinity tool. Apilimod is a potent small molecular inhibitor of IL-12/IL-23 with an unknown target and has been evaluated in clinical trials for patients with Crohn's disease or rheumatoid arthritis. Using a chemical genetic approach, we show that it binds to PIKfyve and blocks its phosphotransferase activity, leading to selective inhibition of IL-12/IL-23p40. Pharmacological or genetic inactivation of PIKfyve is necessary and sufficient for suppression of IL-12/IL-23p40 expression. Thus, we have uncovered a phosphoinositide-mediated regulatory mechanism that controls TLR signaling.

Structure of human tankyrase 1 in complex with small-molecule inhibitors PJ34 and XAV939.

The crystal structures of tankyrase 1 (TNKS1) in complex with two small-molecule inhibitors, PJ34 and XAV939, both at 2.0 Å resolution, are reported. The structure of TNKS1 in complex with PJ34 reveals two molecules of PJ34 bound in the NAD(+) donor pocket. One molecule is in the nicotinamide portion of the pocket, as previously observed in other PARP structures, while the second molecule is bound in the adenosine portion of the pocket. Additionally, unlike the unliganded crystallization system, the TNKS1-PJ34 crystallization system has the NAD(+) donor site accessible to bulk solvent in the crystal, which allows displacement soaking. The TNKS1-PJ34 crystallization system was used to determine the structure of TNKS1 in complex with XAV939. These structures provide a basis for the start of a structure-based drug-design campaign for TNKS1.

Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling.

The stability of the Wnt pathway transcription factor beta-catenin is tightly regulated by the multi-subunit destruction complex. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. However, the development of targeted Wnt pathway inhibitors has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. Here, we used a chemical genetic screen to identify a small molecule, XAV939, which selectively inhibits beta-catenin-mediated transcription. XAV939 stimulates beta-catenin degradation by stabilizing axin, the concentration-limiting component of the destruction complex. Using a quantitative chemical proteomic approach, we discovered that XAV939 stabilizes axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2. Both tankyrase isoforms interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin-proteasome pathway. Thus, our study provides new mechanistic insights into the regulation of axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies.

Diastereoselective metal-catalyzed [4 + 2 + 2] carbocyclization reactions utilizing a rhodium N-heterocyclic carbene (NHC) complex: the first example of a rhodium NHC-catalyzed [m + n + o] carbocyclization.

The air and moisture stable rhodium N-heterocyclic carbene (NHC) complex, RhCl(IMes)(COD)(IMes =N,N[prime or minute]-bis(2,4,6-trimethylphenyl)imidazole-2-ylidine; COD = 1,5-cyclooctadiene), facilitates a diastereoselective metal-catalyzed [4 + 2 + 2] carbocyclization of 1,6-enynes in the presence of silver triflate and 1,3-butadiene.

Kinetic and docking studies of the interaction of quinones with the quinone reductase active site.

NAD(P)H/quinone acceptor oxidoreductase type 1 (QR1) protects cells from cytotoxic and neoplastic effects of quinones though two-electron reduction. Kinetic experiments, docking, and binding affinity calculations were performed on a series of structurally varied quinone substrates. A good correlation between calculated and measured binding affinities from kinetic determinations was obtained. The experimental and theoretical studies independently support a model in which quinones (with one to three fused aromatic rings) bind in the QR1 active site utilizing a pi-stacking interaction with the isoalloxazine ring of the FAD cofactor.

Intermolecular transition metal-catalyzed [4 + 2 + 2] cycloaddition reactions: a new approach to the construction of eight-membered rings.

Transition metal-catalyzed cycloaddition reactions represent powerful methods for the construction of complex polycyclic systems. We have developed a new intermolecular metal-catalyzed [4 + 2 + 2] cycloaddition of heteroatom-tethered enyne derivatives with 1,3-butadiene. This study demonstrates that excellent selectivity can be obtained for the heterocycloaddition adducts through the judicious choice of silver salt. The development of the tandem rhodium-catalyzed allylic substitution [4 + 2 + 2] cycloaddition provides a convenient three-component coupling that circumvents the prior formation of the enyne derivative. Finally, the introduction of a stereogenic center at C-2 leads to a diastereoselective cycloaddition, which provides a powerful new method for the construction of bicyclic octanoid ring systems applicable to target directed synthesis.

Regioselective organocadmium alkylations of substituted quinones.

A series of substituted quinones was alkylated with diethylcadmium. Regiochemistry of addition shifted from quinol formation to conjugate addition as a function of the steric and electronic effects of the substituents.