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CDK7 has emerged as an exciting target in oncology due to its roles in two important processes that are misregulated in cancer cells: cell cycle and transcription. This report describes the discovery of SY-5609, a highly potent (sub-nM CDK7 Kd) and selective, orally available inhibitor of CDK7 that entered the clinic in 2020 (ClinicalTrials.gov Identifier: NCT04247126). Structure-based design was leveraged to obtain high selectivity (>4000-times the closest off target) and slow off-rate binding kinetics desirable for potent cellular activity. Finally, incorporation of a phosphine oxide as an atypical hydrogen bond acceptor helped provide the required potency and metabolic stability. The development candidate SY-5609 displays potent inhibition of CDK7 in cells and demonstrates strong efficacy in mouse xenograft models when dosed as low as 2 mg/kg.

Recent studies suggest that targeting transcriptional machinery can lead to potent and selective anticancer effects in cancers dependent on high and constant expression of certain transcription factors for growth and survival. Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the CDK-activating kinase complex. Its function is required for both cell-cycle regulation and transcriptional control of gene expression. CDK7 has recently emerged as an attractive cancer target because its inhibition leads to decreased transcript levels of oncogenic transcription factors, especially those associated with super-enhancers. Here, we describe a selective CDK7 inhibitor SY-1365, which is currently in clinical trials in populations of patients with ovarian and breast cancer (NCT03134638). In vitro, SY-1365 inhibited cell growth of many different cancer types at nanomolar concentrations. SY-1365 treatment decreased MCL1 protein levels, and cancer cells with low BCL2L1 (BCL-XL) expression were found to be more sensitive to SY-1365. Transcriptional changes in acute myeloid leukemia (AML) cell lines were distinct from those following treatment with other transcriptional inhibitors. SY-1365 demonstrated substantial antitumor effects in multiple AML xenograft models as a single agent; SY-1365–induced growth inhibition was enhanced in combination with the BCL2 inhibitor venetoclax. Antitumor activity was also observed in xenograft models of ovarian cancer, suggesting the potential for exploring SY-1365 in the clinic in both hematologic and solid tumors. Our findings support targeting CDK7 as a new approach for treating transcriptionally addicted cancers. Significance: These findings demonstrate the molecular mechanism of action and potent antitumor activity of SY-1365, the first selective CDK7 inhibitor to enter clinical investigation.

CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a “master regulator” role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.

The amine-catalyzed epimerization of chiral γ-hydroxybutenolides leads to a fast exchange on the NMR time scale.

CDK12 and CDK13 regulate expression of large transcripts requiring substantial processing to produce mature mRNA. This transcriptional regulation includes coordinated phosphorylation of specific repeats within the C-terminal domain of RNA polymerase II and association with RNA processing factors (Chila, 2016). RNAi knockdown of CDK12 in cell culture decreases expression of DNA damage response genes, including BRCA1 and ATR, while enhancing sensitivity to DNA damaging agents (Blazek, 2011; Liang, 2015). Recently THZ531, a selective covalent inhibitor of CDK12 and CDK13, was shown to decrease expression of DNA damage response genes in cell culture (Zhang, 2016). Here we present further studies with THZ531 to guide our discovery program toward molecules suitable for clinical development and to explore mechanistic rationales for combining a CDK12/13 inhibitor with PARP inhibitors or DNA damaging agents for difficult-to-treat cancers such as high-grade serous ovarian cancer and triple-negative breast cancer. Using THZ531 as a benchmark, we developed assays capable of discriminating sub-nM inhibitors, including quantifying time-dependent covalent inhibition and cell-based CDK occupancy. Since CDK7, like CDK12 and CDK13, contains a cysteine residue proximal to the kinase active site, these approaches are critical to understand covalent inhibitor selectivity. Furthermore, we performed kinome paneling studies to better understand selectivity of this scaffold in support of our ongoing efforts to optimize CDK12/13 potency and selectivity. To pharmacologically investigate the previously reported effects of CDK12 RNAi, growth inhibition of a panel of ovarian and breast cancer cell lines was assessed following treatment with THZ531 (OVA EC50 = 50-200 nM (n=6); BRCA EC50 400) to reveal relationships between inhibitor sensitivity, mutation status, gene expression, and potential oncology indications that may be addressed by these different mechanisms. Finally THZ531 was synergistic with both PARP inhibitors and DNA damaging agents in ovarian and breast cancer cell lines. These data highlight cancer indications and combinations that may be particularly amenable to treatment with CDK12/13 inhibitors. While the pharmacokinetic properties of THZ531 preclude adequate target engagement in tumor tissue at tolerated doses in mouse model systems, our ongoing medicinal chemistry program is progressing to identify and optimize CDK12/13 inhibitors suitable for clinical evaluation. Citation Format: Michael Bradley, Jason Marineau, Yoon Choi, Kristin Hamman, Goran Malojcic, David Orlando, Yixuan Ren, Nan Ke, Shanhu Hu, Eric Olson, Christian Fritz, Christopher Roberts. Targeting the transcriptional kinases CDK12 and CDK13 in breast and ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1143. doi:10.1158/1538-7445.AM2017-1143

The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway essential for cancer cell growth and survival. MLN4924 is a selective inhibitor of NAE currently in clinical trials for the treatment of cancer. Here, we show that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by MLN4924. Importantly, we have determined that compounds resembling MLN4924 demonstrate the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) catalyzed by their cognate-activating enzymes. These findings reveal insights into the mechanism of E1s and suggest a general strategy for selective inhibition of UBL conjugation pathways.