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Detail description of synthesis and supporting data

Figure S1. In vitro profiling of SCH900776 and its selected analogs. Figure S2. Analogs of MU380 with the N-trifluoromethylpyrazol pharmacophore. Figure S3. Dot plots of flow cytometric analysis using γH2AX and Annexin V staining related to Figure 2D. Figure S4. Dose response curves of relative viability of the tested cell lines treated with CHK1 inhibitors at indicated concentrations in combination with HU in the concentration range shown on the x-axis (related to Figure 3A). Figure S5. Dynamics of cytotoxic effects of CHK1 inhibitors alone and in combination with HU. Figure S6. Dose response curves of relative viability of the tested cell lines treated with CHK1 inhibitors at indicated concentration in combination with GEM in concentration range shown on the x-axis (related to Figure 3B). Figure S7. Dose response curves of relative viability of the tested cell lines treated with CHK1 inhibitors at indicated concentration in combination with irradiation in the dose range shown on the x-axis. Figure S8. Western blot analysis of total CHK1 and p-CHK1 (S296) levels in a panel of selected cell lines after indicated treatments. Figure S9. Drug retention analysis. Figure S10. MU380 induces CHK1 pS345 in mice hair follicles. Figure S11. Bioluminescent signal of SHO mice bearing MiaPaCa2 luc xenografts treated with GEM, SCH900776, or MU380 alone or in indicated combinations. Weight of MiaPaCa2 luc tumor-bearing animals. Representative images of γH2AX staining in MiaPaCa2 luc tumor sections.

Detail description of Material and Methods

Supplementary Legends for Supplemetary Figure S1 - S11

Ariadne is a non-hallucinogenic analog in the phenylalkylamine chemical class of psychedelics that is closely related to an established synthetic hallucinogen, DOM, differing only by one methylene group in the α-position to the amine. Ariadne has been tested in humans including clinical trials at the Bristol-Myers pharmaceutical company that indicate a lack of hallucinogenic effects and remarkable therapeutic effects, such as rapid remission of psychotic symptoms in schizophrenics, relaxation in catatonics, complete remission of symptoms in Parkinson’s disease, and improved pro-cognitive effects in geriatric subjects. Despite these provocative clinical results, the compound has been abandoned as a drug candidate and its molecular pharmacology remained unknown. Here, we report a detailed examination of the in vitro and in vivo pharmacology of Ariadne and its analogs and propose a molecular hypothesis for the lack of hallucinogenic effects and therapeutic potential of this compound class. We also provide a summary of previous clinical and preclinical results to contextualize the molecular signaling data. Our results show that Ariadne is a serotonin 5-HT(2) receptor agonist, exhibits modest selectivity over 5-HT(1E) and 5-HT(1F) receptors, has no relevant activity at 5-HT(5,6,7) and other aminergic receptors, and no substantial affinity at plasma membrane monoamine transporters. Compared to DOM, Ariadne shows lower signaling potency and efficacy in multiple signaling pathways examined (G(q), G(11), and β-arrestin2) coupled to 5-HT(2A) receptors. We confirmed the shift in signaling for an alpha-propyl analog and provide a molecular docking rationale for the progressive decrease in signaling potency with growing length of the α-substituent. Ariadne versus DOM exhibits no apparent change in the relative preference between G(q/11) activation and β-arrestin2 recruitment, instead there is a small but consistent drop in efficacy in these signaling channels. Ariadne acts as a 5-HT(2A) agonist in vivo in mice and shows markedly attenuated head twitch response (HTR) in comparison to its hallucinogenic analogs, consistent with previous studies in rabbits, cats, and dogs. Hence, we propose the lower 5-HT(2A) receptor signaling efficacy of this compound class as an explanatory model for the lack of hallucinogenic effects of Ariadne in humans, and the dramatically attenuated hallucinosis-like effects in animals (5-HT(2A) signaling efficacy hypothesis). In terms of reverse translation of the noted clinical therapeutic effects, we used an auxilin knock-out model of Parkinson’s disease where Ariadne rescued severe motor deficits in this mouse line, on par to effects of L-DOPA, a notable finding considering Ariadne’s lack of activity at dopamine receptors and transporters. Ariadne emerges as a prototype of a new drug class, non-hallucinogenic 5HT(2A) agonists, with considerable therapeutic potential across psychiatric and neurological indications.

CDK12 is a kinase associated with elongating RNA polymerase II (RNAPII) and is frequently mutated in cancer. CDK12 depletion reduces the expression of homologous recombination (HR) DNA repair genes, but comprehensive insight into its target genes and cellular processes is lacking. We use a chemical genetic approach to inhibit analog‐sensitive CDK12, and find that CDK12 kinase activity is required for transcription of core DNA replication genes and thus for G1/S progression. RNA‐seq and ChIP‐seq reveal that CDK12 inhibition triggers an RNAPII processivity defect characterized by a loss of mapped reads from 3′ends of predominantly long, poly(A)‐signal‐rich genes. CDK12 inhibition does not globally reduce levels of RNAPII‐Ser2 phosphorylation. However, individual CDK12‐dependent genes show a shift of P‐Ser2 peaks into the gene body approximately to the positions where RNAPII occupancy and transcription were lost. Thus, CDK12 catalytic activity represents a novel link between regulation of transcription and cell cycle progression. We propose that DNA replication and HR DNA repair defects as a consequence of CDK12 inactivation underlie the genome instability phenotype observed in many cancers.

The traditional way of rationally engineering enzymes to change their biocatalytic properties utilizes the modifications of their active sites. Another emerging approach is the engineering of structural features involved in the exchange of ligands between buried active sites and the surrounding solvent. However, surprisingly little is known about the effects of mutations that alter the access tunnels on the enzymes’ catalytic properties, and how these tunnels should be redesigned to allow fast passage of cognate substrates and products. Thus, we have systematically studied the effects of single-point mutations in a tunnel-lining residue of a haloalkane dehalogenase on the binding kinetics and catalytic conversion of both linear and branched haloalkanes. The hotspot residue Y176 was identified using computer simulations and randomized through saturation mutagenesis, and the resulting variants were screened for shifts in binding rates. Strikingly, opposite effects of the substituted residues on the catalytic efficiency toward linear and branched substrates were observed, which was found to be due to substrate-specific requirements in the critical steps of the respective catalytic cycles. We conclude that not only the catalytic sites, but also the access pathways must be tailored specifically for each individual ligand, which is a new paradigm in protein engineering and de novo protein design. A rational approach is proposed here to address more effectively the task of designing ligand-specific tunnels using computational tools.