Design principles for cyclin K molecular glue degraders.

Molecular glue degraders are an effective therapeutic modality, but their design principles are not well understood. Recently, several unexpectedly diverse compounds were reported to deplete cyclin K by linking CDK12-cyclin K to the DDB1-CUL4-RBX1 E3 ligase. Here, to investigate how chemically dissimilar small molecules trigger cyclin K degradation, we evaluated 91 candidate degraders in structural, biophysical and cellular studies and reveal all compounds acquire glue activity via simultaneous CDK12 binding and engagement of DDB1 interfacial residues, in particular Arg928. While we identify multiple published kinase inhibitors as cryptic degraders, we also show that these glues do not require pronounced inhibitory properties for activity and that the relative degree of CDK12 inhibition versus cyclin K degradation is tuneable. We further demonstrate cyclin K degraders have transcriptional signatures distinct from CDK12 inhibitors, thereby offering unique therapeutic opportunities. The systematic structure-activity relationship analysis presented herein provides a conceptual framework for rational molecular glue design.

Overlaid transcriptome and proteome analyses identify mitotic kinesins as important targets of arylsulfonamide-mediated RBM39 degradation.

Certain arylsulfonamides (ArSulfs) induce an interaction between the E3 ligase substrate adaptor DCAF15 and the critical splicing factor RBM39, ultimately causing its degradation. However, degradation of a splicing factor introduces complex pleiotropic effects that are difficult to untangle, since, aside from direct protein degradation, downstream transcriptional effects also influence the proteome. By overlaying transcriptional data and proteome datasets, we distinguish transcriptional from direct degradation effects, pinpointing those proteins most impacted by splicing changes. Using our workflow, we identify and validate the upregulation of the argininie-and-serine rich protein (RSRP1) and the downregulation of the key kinesin motor proteins KIF20A and KIF20B due to altered splicing in the absence of RBM39. We further show that kinesin downregulation is connected to the multinucleation phenotype observed upon RBM39 depletion by ArSulfs. Our approach should be helpful in the assessment of potential cancer drug candidates which target splicing factors. Implications: Our approach provides a workflow for identifying and studying the most strongly modulated proteins when splicing is altered; the work also uncovers a splicing-based approach toward pharmacological targeting of mitotic kinesins.

Overlaid Transcriptional and Proteome Analyses Identify Mitotic Kinesins as Important Targets of Arylsulfonamide-Mediated RBM39 Degradation.

Certain arylsulfonamides (ArSulf) induce an interaction between the E3 ligase substrate adaptor DCAF15 and the critical splicing factor RBM39, ultimately causing its degradation. However, degradation of a splicing factor introduces complex pleiotropic effects that are difficult to untangle, since, aside from direct protein degradation, downstream transcriptional effects also influence the proteome. By overlaying transcriptional data and proteome datasets, we distinguish transcriptional from direct degradation effects, pinpointing those proteins most impacted by splicing changes. Using our workflow, we identify and validate the upregulation of the arginine-and-serine rich protein (RSRP1) and the downregulation of the key kinesin motor proteins KIF20A and KIF20B due to altered splicing in the absence of RBM39. We further show that kinesin downregulation is connected to the multinucleation phenotype observed upon RBM39 depletion by ArSulfs. Our approach should be helpful in the assessment of potential cancer drug candidates which target splicing factors. IMPLICATIONS: Our approach provides a workflow for identifying and studying the most strongly modulated proteins when splicing is altered. The work also uncovers a splicing-based approach toward pharmacologic targeting of mitotic kinesins.

Divergent Synthesis of Bioactive Dithiodiketopiperazine Natural Products Based on a Double C(sp3 )-H Activation Strategy.

This article provides a detailed report of our efforts to synthesize the dithiodiketopiperazine (DTP) natural products (-)-epicoccin G and (-)-rostratin A using a double C(sp3 )-H activation strategy. The strategy's viability was first established on a model system lacking the C8/C8' alcohols. Then, an efficient stereoselective route including an organocatalytic epoxidation was secured to access a key bis-triflate substrate. This bis-triflate served as the functional handles for the key transformation of the synthesis: a double C(sp3 )-H activation. The successful double activation opened access to a common intermediate for both natural products in high overall yield and on a multigram scale. After several unsuccessful attempts, this intermediate was efficiently converted to (-)-epicoccin G and to the more challenging (-)-rostratin A via suitable oxidation/reduction and protecting group sequences, and via a final sulfuration that occurred in good yield and high diastereoselectivity. These efforts culminated in the synthesis of (-)-epicoccin G and (-)-rostratin A in high overall yields (19.6 % over 14 steps and 12.7 % over 17 steps, respectively), with the latter being obtained on a 500 mg scale. Toxicity assessments of these natural products and several analogues (including the newly synthesized epicoccin K) in the leukemia cell line K562 confirmed the importance of the disulfide bridge for activity and identified dianhydrorostratin A as a 20x more potent analogue.