Development of Highly Potent and Selective Steroidal Inhibitors and Degraders of CDK8

Cortistatin A is a natural product isolated from the marine sponge Corticium simplex and was found to be a potent and selective inhibitor of CDK8. Many synthetic groups have reported total syntheses of Cortistatin A; however, these syntheses require between 16 and 30 steps and report between 0.012–2% overall yields, which is not amenable to large-scale production. Owing to similarities between the complex core of Cortistatin A and the simple steroid core, we initiated a campaign to design simple, more easily prepared CDK8 inhibitors based on a steroid scaffold that would be more convenient for large-scale synthesis. Herein, we report the discovery and optimization of JH-VIII-49, a potent and selective inhibitor of CDK8 with a simple steroid core that has an eight-step synthesis with a 33% overall yield, making it suitable for large-scale preparation. Using this scaffold, we then developed a bivalent small molecule degrader, JH-XI-10-02, that can recruit the E3 ligase CRL4^Cereblon to promote the ubiquitination and proteosomal degradation of CDK8

In vitro inhibitor specificity profiles with recombinant Fes and Flt3 kinases.

<p>In vitro kinase assays were performed with recombinant full-length Fes as well as wild-type (WT), ITD and D835Y forms of the Flt3 kinase domain (Z’-Lyte assay; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181178#sec002" target="_blank">Materials and methods</a>). Each inhibitor was tested in triplicate over a range of concentrations with the ATP concentration set to the K_m value for each kinase. IC₅₀ values were calculated by non-linear regression analysis of the resulting dose-response curves. Also included in the table are the IC₅₀ values for growth suppression by each compound in four AML cell lines, calculated from the dose-response curves shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181178#pone.0181178.g002" target="_blank">Fig 2</a>. Note that MV4-11, MOLM-13, and MOLM-14 are Flt3-ITD⁺, while THP-1 is wild-type for Flt3.</p

Identification and attempted validation of BAY61-3606 targets.

<p>(<b>a</b>) TREEspot image representing the inhibitory activity of BAY61-3606. Kinases that are inhibited for ATP binding by BAY61-3606 are indicated by red dots on the phylogenetic tree of kinases. The size of the red dot corresponds to the amount of inhibition by 1 μM BAY61-3606. The identity of the individual kinases can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041343#pone.0041343.s008" target="_blank">Table S1</a>. (<b>b</b>) Cell viability quantified by Syto60 after shRNA-mediated knockdown of the potential BAY61-3606 targets in DLD-1 (red) or DKs-8 (blue) cell lines. Relative cell viability is normalized to the parental cell lines infected with vector only. Error bars represent SEM for 2 independent experiments.</p

MAP4K2 modulates NFκβ signaling.

<p>(<b>a</b>) Time course of phospho-Iκβα (Ser32/Ser36) activation after 1 µM AZ-628 treatment in HCT-116 (red lines) or HKe-3 (blue line) cells with MAP4K2 knock down, as measured via Bio-Plex. Relative signal was normalized to a master control lysate. Error bars represent SEM for 3 independent experiments. NFκβ signaling was enhanced in HKe-3 cells after exposure to AZ-628 and this was dependent upon MAP4K2. (<b>b</b>) Cell viability quantified by Syto60 after 72 hours of combinatorial treatment with IKK inhibitor VII and 1 µM AZ-628. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Like BAY61-3606, IKK inhibitor VII enhanced the effect of AZ-628 specifically in K-RAS wild-type cells. (<b>c</b>) Cell viability after 72 hours of combinatorial treatment of 1 µM IKK inhibitor VII and 1 µM AZ-628 (shaded) or 1 µM AZ-628 alone (clear) in HKe3 cells with MAP4K2 knockdown. Relative cell viability was normalized to 1 µM AZ-628 treated samples. Loss of MAP4K2 abrogated the ability of IKK inhibitor VII to sensitize HKe-3 cells to AZ-628.</p

Inhibition of SYK is not responsible for the BAY61-3606 effect on cell viability in colorectal cancer cells.

<p>(<b>a</b>) Knockdown of SYK protein in DLD-1 cells via shRNA, as shown by Western Blotting. The Ramos (R) cell line, a hematopoetic cell line with a high expression of SYK, was used as a positive control. (<b>b</b>) Cell viability of DLD-1 cells (red), and its K-RAS wild-type (DKs-8 – blue) and SYK knockdown (red shaded) derivatives after 72 hours of treatment with 1 <b>μ</b>M of BAY61-3606 or R406, a distinct SYK inhibitor. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Error bars represent SEM for 3 independent experiments. DLD-1 cells and its K-RAS wild-type derivative did not exhibit sensitivity to R406, while knocking down SYK in DLD-1 cells only minimally affected its sensitivity to BAY61-3606.</p

BAY61-3606 affects viability in cells expressing mutant K-RAS or B-RAF through a MAPK-independent pathway.

<p>(<b>a</b>) Cell viability quantified by Syto60 after 72 hours of AZ-628, CI-1040 or BAY61-3606 treatment in HCT-116 (K-RAS^G13D/+, red) or HKe-3 (K-RAS^−/+, blue) cell lines. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Error bars represent SEM for 3 independent experiments. Cells expressing mutant K-RAS were relatively sensitive to AZ-628 and BAY61-3606, but not CI-1040. (<b>b</b>) Cell cycle profiles, as determined by propidium iodide staining, of colorectal cancer cells with mutant B-RAF (HT-29) treated with CI-1040 or mutant K-RAS (DLD-1) treated with BAY61-3606. While inhibition of MEK induces G1 arrest in HT-29 cells, as evidenced by the loss of the 4N peak, BAY61-3606 did not appear to alter the profile of DLD-1 cells. (<b>c</b>) Cell viability of colorectal cancer cells expressing mutant B-RAF (V600E) after 72 hours of treatment with BAY61-3606 and/or CI-1040. Cells expressing mutant B-RAF (HT-29 – solid outline and RKO – dotted outline) were sensitive to both BAY61-3606 and CI-1040 and these two inhibitors cooperated to produce an enhanced response. (<b>d</b>) Phospho-MEK1 (Ser217/Ser221) and phospho-ERK1/2 (Thr202/Tyr204, Thr185/Tyr187) levels in K-RAS wild-type and mutant cells after 45 minute exposure to 1 µM AZ-628, 1 µM BAY61-3606, or vehicle control. Signals were measured using Bio-Plex assays. Relative signal was normalized to a master control lysate. AZ-628 treatment reduced the level of phospho-MEK and phospho-ERK, but BAY61-3606 did not.</p

MAP4K2 is a target for BAY61-3606 that modulates the response of wild-type cells to AZ-628.

<p>(<b>a</b>) Cell viability quantified by Syto60 after 72 hours of combinatorial treatment with varying concentrations of BAY61-3606 and 1 µM AZ-628 in HCT-116 (red line) or HKe-3 (blue lines) cells. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Error bars represent SEM for 3 independent experiments. The two inhibitors cooperate in wild-type cells, but not in cells expressing mutant K-RAS. (<b>b</b>) Cell viability after 72 hours of combinatorial treatment with varying concentrations of BAY derivative 6 and 1 µM AZ-628 in HCT-116 and HKe-3 cells. BAY derivative 6 does not confer additional sensitivity to AZ-628 upon HKe-3 cells. (<b>c</b>) Cell viability quantified by Syto60 after 72 hours of AZ-628 treatment in HCT-116 or HKe-3 cell lines with MAP4K2 knockdown. Loss of MAP4K2 does not affect AZ-628 response in cells expressing mutant K-RAS, but enhances the effect of AZ-628 in cells expressing wild-type K-RAS. (<b>d</b>) Cell viability after 72 hours of combinatorial treatment with 1 µM BAY61-3606 and 1 µM AZ-628 (shaded) or 1 µM AZ-628 alone (clear) in HKe-3 cells with MAP4K2 knockdown. Relative cell viability was normalized to 1 µM AZ-628 treated samples. In parental HKe-3 cells, BAY61-3606 confers sensitivity to AZ-628. Upon loss of MAP4K2, BAY61-3606 no longer sensitizes.</p

Activity of BAY61-3606 against selected kinases.

*<p>% inhibition of ATP binding as assessed by KINOMEscan™ (Ambit Biosciences).</p>**<p><i>In vitro</i> IC50 were determined by SelectScreen® (Invitrogen).</p

Active mutants of Flt3 (ITD and D835Y) transform human TF-1 cells to cytokine independent growth.

<p>A) The human myeloid cell line TF-1 was transduced with recombinant retroviruses carrying wild-type (WT), ITD, or D835Y forms of Flt3, or with an empty vector as control. Following selection with G418, each of the TF-1 cell populations was cultured in the absence of GM-CSF, and viable cell outgrowth was monitored daily by CellTiter-Blue assay for 4 days. Each population was assayed in triplicate, and results are presented as the mean value ± the SD. B) Fes and Flt3 kinase proteins were immunoprecipitated from each of the cell populations shown in part A, followed by immunoblotting with antibodies to phosphotyrosine (pTyr), the Fes activation loop phosphotyrosine (pY713), as well as the Flt3 and Fes proteins. This experiment was performed in triplicate with comparable results, and a representative example is shown.</p

HG7-92-01 treatment induces apoptosis in patient-derived Flt3-ITD⁺ AML bone marrow cells.

<p>Cryopreserved biopsy samples from twelve randomly chosen Flt3-ITD⁺ AML patients (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181178#pone.0181178.t003" target="_blank">Table 3</a>) were thawed and cultured on mitotically inactivated Hs27 feeder fibroblasts in the presence of myeloid cytokines as described in the text. After 24 h in culture, cells were treated with HG7-92-01 or tandutinib at 1 and 3 μM or with the DMSO carrier solvent as negative control. The presence of apoptotic cells was then assayed 48 and 72 h later by flow cytometry for cell-surface phosphatidylserine. Data are expressed as the increase in percentage of apoptotic cells observed at each time point relative to the DMSO control. To assess non-specific cytotoxicity, bone marrow mononuclear cells from three normal donors were cultured and treated with inhibitors under identical conditions, followed by flow cytometry for apoptotic cells. Characteristics of these three donors are as follows: normal bone marrow donor 1 (NBM1), female, 40 years; NBM2, male, 34 years; NBM3, male, 45 years.</p