PROTACs Targeting BRM (SMARCA2) Afford Selective In Vivo Degradation over BRG1 (SMARCA4) and Are Active in BRG1 Mutant Xenograft Tumor Models.

The identification of VHL-binding proteolysis targeting chimeras (PROTACs) that potently degrade the BRM protein (also known as SMARCA2) in SW1573 cell-based experiments is described. These molecules exhibit between 10- and 100-fold degradation selectivity for BRM over the closely related paralog protein BRG1 (SMARCA4). They also selectively impair the proliferation of the H1944 "BRG1-mutant" NSCLC cell line, which lacks functional BRG1 protein and is thus highly dependent on BRM for growth, relative to the wild-type Calu6 line. In vivo experiments performed with a subset of compounds identified PROTACs that potently and selectively degraded BRM in the Calu6 and/or the HCC2302 BRG1 mutant NSCLC xenograft models and also afforded antitumor efficacy in the latter system. Subsequent PK/PD analysis established a need to achieve strong BRM degradation (>95%) in order to trigger meaningful antitumor activity in vivo. Intratumor quantitation of mRNA associated with two genes whose transcription was controlled by BRM (PLAU and KRT80) also supported this conclusion.

Scaffold-Hopping Approach To Discover Potent, Selective, and Efficacious Inhibitors of NF-κB Inducing Kinase.

NF-κB-inducing kinase (NIK) is a protein kinase central to the noncanonical NF-κB pathway downstream from multiple TNF receptor family members, including BAFF, which has been associated with B cell survival and maturation, dendritic cell activation, secondary lymphoid organ development, and bone metabolism. We report herein the discovery of lead chemical series of NIK inhibitors that were identified through a scaffold-hopping strategy using structure-based design. Electronic and steric properties of lead compounds were modified to address glutathione conjugation and amide hydrolysis. These highly potent compounds exhibited selective inhibition of LTßR-dependent p52 translocation and transcription of NF-κB2 related genes. Compound 4f is shown to have a favorable pharmacokinetic profile across species and to inhibit BAFF-induced B cell survival in vitro and reduce splenic marginal zone B cells in vivo.

An inhibitor's-eye view of the ATP-binding site of CDKs in different regulatory states.

We have used a chemically diverse panel of kinase inhibitors to assess the chemical similarity of the ATP-binding sites of cyclin-dependent kinase (CDK) subfamily members in a range of activation states. Using this approach, we find that different activation states of a particular CDK may differ from each other as much as different CDKs in the same activation state. We also find that inhibitors discriminate more effectively among CDK family members in their monomeric state than in their cyclin-bound state, providing direct evidence for the belief that selective binding to inactive kinase states might be more readily achieved than selective binding to active states.

Comparative structural and functional studies of 4-(thiazol-5-yl)-2-(phenylamino)pyrimidine-5-carbonitrile CDK9 inhibitors suggest the basis for isotype selectivity.

Cyclin-dependent kinase 9/cyclin T, the protein kinase heterodimer that constitutes positive transcription elongation factor b, is a well-validated target for treatment of several diseases, including cancer and cardiac hypertrophy. In order to aid inhibitor design and rationalize the basis for CDK9 selectivity, we have studied the CDK-binding properties of six different members of a 4-(thiazol-5-yl)-2-(phenylamino)pyrimidine-5-carbonitrile series that bind to both CDK9/cyclin T and CDK2/cyclin A. We find that for a given CDK, the melting temperature of a CDK/cyclin/inhibitor complex correlates well with inhibitor potency, suggesting that differential scanning fluorimetry (DSF) is a useful orthogonal measure of inhibitory activity for this series. We have used DSF to demonstrate that the binding of these compounds is independent of the presence or absence of the C-terminal tail region of CDK9, unlike the binding of the CDK9-selective inhibitor 5,6-dichlorobenzimidazone-1-ß-d-ribofuranoside (DRB). Finally, on the basis of 11 cocrystal structures bound to CDK9/cyclin T or CDK2/cyclin A, we conclude that selective inhibition of CDK9/cyclin T by members of the 4-(thiazol-5-yl)-2-(phenylamino)pyrimidine-5-carbonitrile series results from the relative malleability of the CDK9 active site rather than from the formation of specific polar contacts.

Substituted 4-(thiazol-5-yl)-2-(phenylamino)pyrimidines are highly active CDK9 inhibitors: synthesis, X-ray crystal structures, structure-activity relationship, and anticancer activities.

Cancer cells often have a high demand for antiapoptotic proteins in order to resist programmed cell death. CDK9 inhibition selectively targets survival proteins and reinstates apoptosis in cancer cells. We designed a series of 4-thiazol-2-anilinopyrimidine derivatives with functional groups attached to the C5-position of the pyrimidine or to the C4-thiazol moiety and investigated their effects on CDK9 potency and selectivity. One of the most selective compounds, 12u inhibits CDK9 with IC(50) = 7 nM and shows over 80-fold selectivity for CDK9 versus CDK2. X-ray crystal structures of 12u bound to CDK9 and CDK2 provide insights into the binding modes. This work, together with crystal structures of selected inhibitors in complex with both enzymes described in a companion paper, (34) provides a rationale for the observed SAR. 12u demonstrates potent anticancer activity against primary chronic lymphocytic leukemia cells with a therapeutic window 31- and 107-fold over those of normal B- and T-cells.

The CDK9 C-helix exhibits conformational plasticity that may explain the selectivity of CAN508.

CDK9 is the kinase of positive transcription elongation factor b and facilitates the transition of paused RNA polymerase II to processive transcription elongation. CDK9 is a validated target for the treatment of cancer, cardiac hypertrophy, and human immunodeficiency virus. Here we analyze different CDK9/cyclin T variants to identify a form of the complex amenable to use in inhibitor design. To demonstrate the utility of this system, we have determined the crystal structures of CDK9/cyclin T and CDK2/cyclin A bound to the CDK9-specific inhibitor CAN508. Comparison of the structures reveals CDK9-specific conformational changes and identifies a CDK9-specific hydrophobic pocket, adjacent to the αC-helix. By comparison with a previously published structure of CDK9/cyclin T/human immunodeficiency virus TAT we find that the CDK9 αC-helix has a degree of conformational variability that has the potential to be exploited for inhibitor design.

The CDK9 tail determines the reaction pathway of positive transcription elongation factor b.

CDK9, the kinase of positive transcription elongation factor b (P-TEFb), stimulates transcription elongation by phosphorylating RNA polymerase II and transcription elongation factors. Using kinetic analysis of a human P-TEFb complex consisting of CDK9 and cyclin T, we show that the CDK9 C-terminal tail sequence is important for the catalytic mechanism and imposes an ordered binding of substrates and release of products. Crystallographic analysis of a CDK9/cyclin T complex in which the C-terminal tail partially blocks the ATP binding site reveals a possible reaction intermediate. Biochemical characterization of CDK9 mutants supports a model in which the CDK9 tail cycles through different conformational states. We propose that this mechanism is critical for the pattern of CTD Ser2 phosphorylation on actively transcribed genes.

CDK Inhibitors Roscovitine and CR8 Trigger Mcl-1 Down-Regulation and Apoptotic Cell Death in Neuroblastoma Cells.

Neuroblastoma (NB), the most frequent extracranial solid tumor of children accounting for nearly 15% of all childhood cancer mortality, displays overexpression of antiapoptotic Bcl-2 and Mcl-1 in aggressive forms of the disease. The clinical phase 2 drug roscovitine (CYC202, seliciclib), a relatively selective inhibitor of cyclin-dependent kinases (CDKs), and CR8, a recently developed and more potent analog, induce concentration-dependent apoptotic cell death of NB cells (average IC(50) values: 24.2 µM and 0.4 µM for roscovitine and CR8, respectively). Both roscovitine and CR8 trigger rapid down-regulation of the short-lived survival factor Mcl-1 in the 9 investigated human NB cell lines. This effect was further analyzed in the human SH-SY5Y NB cell line. Down-regulation of Mcl-1 appears to depend on inhibition of CDKs rather than on interaction of roscovitine and CR8 with their secondary targets. CR8 is an adenosine triphosphate-competitive inhibitor of CDK9, and the structure of a CDK9/cyclin T/CR8 complex is described. Mcl-1 down-regulation occurs both at the mRNA and protein levels. This effect can be accounted for by a reduction in Mcl-1 protein synthesis, under stable Mcl-1 degradation conditions. Mcl-1 down-regulation is accompanied by a transient increase in free Noxa, a proapoptotic factor. Mcl-1 down-regulation occurs independently of the presence or up-regulation of p53 and of the MYCN status. Taken together, these results suggest that the clinical drug roscovitine and its novel analog CR8 induce apoptotic tumor cell death by down-regulating Mcl-1, a key survival factor expressed in all NB cell lines. CDK inhibition may thus constitute a new approach to treat refractory high-risk NB.