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Histone methyltransferase DOT1L is an attractive therapeutic target for the treatment of hematological malignancies. Here, we report the design, synthesis, and profiling of new DOT1L inhibitors based on nonroutine carbocyclic C-nucleoside scaffolds. The experimentally observed SAR was found to be nontrivial as seemingly minor changes of individual substituents resulted in significant changes in the affinity to DOT1L. Molecular modeling suggested that these trends could be related to significant conformational changes of the protein upon interaction with the inhibitors. The compounds

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

Kinetin (N6-furfuryladenine), a plant growth substance of the cytokinin family, has been shown to modulate aging and various age-related conditions in animal models. Here we report the synthesis of kinetin isosteres with the purine ring replaced by other bicyclic heterocycles, and the biological evaluation of their activity in several in vitro models related to neurodegenerative diseases. Our findings indicate that kinetin isosteres protect Friedreich́s ataxia patient-derived fibroblasts against glutathione depletion, protect neuron-like SH-SY5Y cells from glutamate-induced oxidative damage, and correct aberrant splicing of the ELP1 gene in fibroblasts derived from a familial dysautonomia patient. Although the mechanism of action of kinetin derivatives remains unclear, our data suggest that the cytoprotective activity of some purine isosteres is mediated by their ability to reduce oxidative stress. Further, the studies of permeation across artificial membrane and model gut and blood-brain barriers indicate that the compounds are orally available and can reach central nervous system. Overall, our data demonstrate that isosteric replacement of the kinetin purine scaffold is a fruitful strategy for improving known biological activities of kinetin and discovering novel therapeutic opportunities.

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.

A series of 6-tosyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide analogues are synthesized by conventional techniques and characterized by elemental analysis, IR, MS, (1)H, and (13)C NMR. These are tested for their antibacterial activity against Bacillus subtilis (abbreviated as BS), Staphylococcus aureus (abbreviated as SA), and Escherichia coli (abbreviated as EC). The synthesized compounds are able to inhibit the growth of the SA and EC. None of the compounds are effective against BS. All valence molecular orbital (abbreviated as MO) calculations with PM6 have been carried out for the molecules for which bioactivity data are available. Ciprofloxacin is taken as the standard antibiotics to compare activity with the molecules synthesized. It has been attempted to correlate the activity of the molecules with their electronic structure.