Targeting transcription in heart failure via CDK7/12/13 inhibition.

Heart failure with reduced ejection fraction (HFrEF) is associated with high mortality, highlighting an urgent need for new therapeutic strategies. As stress-activated cardiac signaling cascades converge on the nucleus to drive maladaptive gene programs, interdicting pathological transcription is a conceptually attractive approach for HFrEF therapy. Here, we demonstrate that CDK7/12/13 are critical regulators of transcription activation in the heart that can be pharmacologically inhibited to improve HFrEF. CDK7/12/13 inhibition using the first-in-class inhibitor THZ1 or RNAi blocks stress-induced transcription and pathologic hypertrophy in cultured rodent cardiomyocytes. THZ1 potently attenuates adverse cardiac remodeling and HFrEF pathogenesis in mice and blocks cardinal features of disease in human iPSC-derived cardiomyocytes. THZ1 suppresses Pol II enrichment at stress-transactivated cardiac genes and inhibits a specific pathologic gene program in the failing mouse heart. These data identify CDK7/12/13 as druggable regulators of cardiac gene transactivation during disease-related stress, suggesting that HFrEF features a critical dependency on transcription that can be therapeutically exploited.

Mapping the Degradable Kinome Provides a Resource for Expedited Degrader Development.

Targeted protein degradation (TPD) refers to the use of small molecules to induce ubiquitin-dependent degradation of proteins. TPD is of interest in drug development, as it can address previously inaccessible targets. However, degrader discovery and optimization remains an inefficient process due to a lack of understanding of the relative importance of the key molecular events required to induce target degradation. Here, we use chemo-proteomics to annotate the degradable kinome. Our expansive dataset provides chemical leads for ∼200 kinases and demonstrates that the current practice of starting from the highest potency binder is an ineffective method for discovering active compounds. We develop multitargeted degraders to answer fundamental questions about the ubiquitin proteasome system, uncovering that kinase degradation is p97 dependent. This work will not only fuel kinase degrader discovery, but also provides a blueprint for evaluating targeted degradation across entire gene families to accelerate understanding of TPD beyond the kinome.

Catalytic Domain Plasticity of MKK7 Reveals Structural Mechanisms of Allosteric Activation and Diverse Targeting Opportunities.

MKK7 (MEK7) is a key regulator of the JNK stress signaling pathway and targeting MKK7 has been proposed as a chemotherapeutic strategy. Detailed understanding of the MKK7 structure and factors that affect its activity is therefore of critical importance. Here, we present a comprehensive set of MKK7 crystal structures revealing insights into catalytic domain plasticity and the role of the N-terminal regulatory helix, conserved in all MAP2Ks, mediating kinase activation. Crystal structures harboring this regulatory helix revealed typical structural features of active kinase, providing exclusively a first model of the MAP2K active state. A small-molecule screening campaign yielded multiple scaffolds, including type II irreversible inhibitors a binding mode that has not been reported previously. We also observed an unprecedented allosteric pocket located in the N-terminal lobe for the approved drug ibrutinib. Collectively, our structural and functional data expand and provide alternative targeting strategies for this important MAP2K kinase.

A broad-spectrum antiviral molecule, QL47, selectively inhibits eukaryotic translation.

Small-molecule inhibitors of translation are critical tools to study the molecular mechanisms of protein synthesis. In this study, we sought to characterize how QL47, a host-targeted, small-molecule antiviral agent, inhibits steady-state viral protein expression. We demonstrate that this small molecule broadly inhibits both viral and host protein synthesis and targets a translation step specific to eukaryotic cells. We show that QL47 inhibits protein neosynthesis initiated by both canonical cap-driven and noncanonical initiation strategies, most likely by targeting an early step in translation elongation. Our findings thus establish QL47 as a new small-molecule inhibitor that can be utilized to probe the eukaryotic translation machinery and that can be further developed as a new therapeutic agent.

A kinase-independent role for CDK8 in BCR-ABL1+ leukemia.

Cyclin-dependent kinases (CDKs) are frequently deregulated in cancer and represent promising drug targets. We provide evidence that CDK8 has a key role in B-ALL. Loss of CDK8 in leukemia mouse models significantly enhances disease latency and prevents disease maintenance. Loss of CDK8 is associated with pronounced transcriptional changes, whereas inhibiting CDK8 kinase activity has minimal effects. Gene set enrichment analysis suggests that the mTOR signaling pathway is deregulated in CDK8-deficient cells and, accordingly, these cells are highly sensitive to mTOR inhibitors. Analysis of large cohorts of human ALL and AML patients reveals a significant correlation between the level of CDK8 and of mTOR pathway members. We have synthesized a small molecule YKL-06-101 that combines mTOR inhibition and degradation of CDK8, and induces cell death in human leukemic cells. We propose that simultaneous CDK8 degradation and mTOR inhibition might represent a potential therapeutic strategy for the treatment of ALL patients.

Development of a Selective CDK7 Covalent Inhibitor Reveals Predominant Cell-Cycle Phenotype.

Cyclin-dependent kinase 7 (CDK7) regulates both cell cycle and transcription, but its precise role remains elusive. We previously described THZ1, a CDK7 inhibitor, which dramatically inhibits superenhancer-associated gene expression. However, potent CDK12/13 off-target activity obscured CDK7s contribution to this phenotype. Here, we describe the discovery of a highly selective covalent CDK7 inhibitor. YKL-5-124 causes arrest at the G1/S transition and inhibition of E2F-driven gene expression; these effects are rescued by a CDK7 mutant unable to covalently engage YKL-5-124, demonstrating on-target specificity. Unlike THZ1, treatment with YKL-5-124 resulted in no change to RNA polymerase II C-terminal domain phosphorylation; however, inhibition could be reconstituted by combining YKL-5-124 and THZ531, a selective CDK12/13 inhibitor, revealing potential redundancies in CDK control of gene transcription. These findings highlight the importance of CDK7/12/13 polypharmacology for anti-cancer activity of THZ1 and posit that selective inhibition of CDK7 may be useful for treatment of cancers marked by E2F misregulation.

Development of Dual and Selective Degraders of Cyclin-Dependent Kinases 4 and 6.

Cyclin-dependent kinases 4 and 6 (CDK4/6) are key regulators of the cell cycle, and there are FDA-approved CDK4/6 inhibitors for treating patients with metastatic breast cancer. However, due to conservation of their ATP-binding sites, development of selective agents has remained elusive. Here, we report imide-based degrader molecules capable of degrading both CDK4/6, or selectively degrading either CDK4 or CDK6. We were also able to tune the activity of these molecules against Ikaros (IKZF1) and Aiolos (IKZF3), which are well-established targets of imide-based degraders. We found that in mantle cell lymphoma cell lines, combined IKZF1/3 degradation with dual CDK4/6 degradation produced enhanced anti-proliferative effects compared to CDK4/6 inhibition, CDK4/6 degradation, or IKZF1/3 degradation. In summary, we report here the first compounds capable of inducing selective degradation of CDK4 and CDK6 as tools to pharmacologically dissect their distinct biological functions.

Homolog-Selective Degradation as a Strategy to Probe the Function of CDK6 in AML.

The design of selective small molecules is often stymied by similar ligand binding pockets. Here, we report BSJ-03-123, a phthalimide-based degrader that exploits protein-interface determinants to achieve proteome-wide selectivity for the degradation of cyclin-dependent kinase 6 (CDK6). Pharmacologic CDK6 degradation targets a selective dependency of acute myeloid leukemia cells, and transcriptomics and phosphoproteomics profiling of acute degradation of CDK6 enabled dynamic mapping of its immediate role in coordinating signaling and transcription.

Targeting MYC dependency in ovarian cancer through inhibition of CDK7 and CDK12/13.

High-grade serous ovarian cancer is characterized by extensive copy number alterations, among which the amplification of MYC oncogene occurs in nearly half of tumors. We demonstrate that ovarian cancer cells highly depend on MYC for maintaining their oncogenic growth, indicating MYC as a therapeutic target for this difficult-to-treat malignancy. However, targeting MYC directly has proven difficult. We screen small molecules targeting transcriptional and epigenetic regulation, and find that THZ1 - a chemical inhibiting CDK7, CDK12, and CDK13 - markedly downregulates MYC. Notably, abolishing MYC expression cannot be achieved by targeting CDK7 alone, but requires the combined inhibition of CDK7, CDK12, and CDK13. In 11 patient-derived xenografts models derived from heavily pre-treated ovarian cancer patients, administration of THZ1 induces significant tumor growth inhibition with concurrent abrogation of MYC expression. Our study indicates that targeting these transcriptional CDKs with agents such as THZ1 may be an effective approach for MYC-dependent ovarian malignancies.

Pharmacological perturbation of CDK9 using selective CDK9 inhibition or degradation.

Cyclin-dependent kinase 9 (CDK9), an important regulator of transcriptional elongation, is a promising target for cancer therapy, particularly for cancers driven by transcriptional dysregulation. We characterized NVP-2, a selective ATP-competitive CDK9 inhibitor, and THAL-SNS-032, a selective CDK9 degrader consisting of a CDK-binding SNS-032 ligand linked to a thalidomide derivative that binds the E3 ubiquitin ligase Cereblon (CRBN). To our surprise, THAL-SNS-032 induced rapid degradation of CDK9 without affecting the levels of other SNS-032 targets. Moreover, the transcriptional changes elicited by THAL-SNS-032 were more like those caused by NVP-2 than those induced by SNS-032. Notably, compound washout did not significantly reduce levels of THAL-SNS-032-induced apoptosis, suggesting that CDK9 degradation had prolonged cytotoxic effects compared with CDK9 inhibition. Thus, our findings suggest that thalidomide conjugation represents a promising strategy for converting multi-targeted inhibitors into selective degraders and reveal that kinase degradation can induce distinct pharmacological effects compared with inhibition.

Activation of the p53 Transcriptional Program Sensitizes Cancer Cells to Cdk7 Inhibitors.

Cdk7, the CDK-activating kinase and transcription factor IIH component, is a target of inhibitors that kill cancer cells by exploiting tumor-specific transcriptional dependencies. However, whereas selective inhibition of analog-sensitive (AS) Cdk7 in colon cancer-derived cells arrests division and disrupts transcription, it does not by itself trigger apoptosis efficiently. Here, we show that p53 activation by 5-fluorouracil or nutlin-3 synergizes with a reversible Cdk7as inhibitor to induce cell death. Synthetic lethality was recapitulated with covalent inhibitors of wild-type Cdk7, THZ1, or the more selective YKL-1-116. The effects were allele specific; a CDK7as mutation conferred both sensitivity to bulky adenine analogs and resistance to covalent inhibitors. Non-transformed colon epithelial cells were resistant to these combinations, as were cancer-derived cells with p53-inactivating mutations. Apoptosis was dependent on death receptor DR5, a p53 transcriptional target whose expression was refractory to Cdk7 inhibition. Therefore, p53 activation induces transcriptional dependency to sensitize cancer cells to Cdk7 inhibition.

A UV-Independent Topical Small-Molecule Approach for Melanin Production in Human Skin.

The presence of dark melanin (eumelanin) within human epidermis represents one of the strongest predictors of low skin cancer risk. Topical rescue of eumelanin synthesis, previously achieved in "redhaired" Mc1r-deficient mice, demonstrated significant protection against UV damage. However, application of a topical strategy for human skin pigmentation has not been achieved, largely due to the greater barrier function of human epidermis. Salt-inducible kinase (SIK) has been demonstrated to regulate MITF, the master regulator of pigment gene expression, through its effects on CRTC and CREB activity. Here, we describe the development of small-molecule SIK inhibitors that were optimized for human skin penetration, resulting in MITF upregulation and induction of melanogenesis. When topically applied, pigment production was induced in Mc1r-deficient mice and normal human skin. These findings demonstrate a realistic pathway toward UV-independent topical modulation of human skin pigmentation, potentially impacting UV protection and skin cancer risk.

Structure-Activity Relationship Study of QL47: A Broad-Spectrum Antiviral Agent.

Here we report the structure-activity relationship (SAR) investigations of QL-XII-47 (QL47), a compound that possesses broad-spectrum antiviral activity against dengue virus and other RNA viruses. A medicinal chemistry campaign initiated from QL47, a previously reported covalent BTK inhibitor, to derive YKL-04-085, which is devoid of any kinase activity when screened against a panel of 468 kinases and with improved pharmacokinetic properties. Both QL47 and YKL-04-085 are potent inhibitors of viral translation and exhibit cellular antiviral activity at 35-fold lower concentrations relative to inhibition of host-cell proliferation.

Discovery of host-targeted covalent inhibitors of dengue virus.

We report here on an approach targeting the host reactive cysteinome to identify inhibitors of host factors required for the infectious cycle of Flaviviruses and other viruses. We used two parallel cellular phenotypic screens to identify a series of covalent inhibitors, exemplified by QL-XII-47, that are active against dengue virus. We show that the compounds effectively block viral protein expression and that this inhibition is associated with repression of downstream processes of the infectious cycle, and thus significantly contributes to the potent antiviral activity of these compounds. We demonstrate that QL-XII-47's antiviral activity requires selective, covalent modification of a host target by showing that the compound's antiviral activity is recapitulated when cells are preincubated with QL-XII-47 and then washed prior to viral infection and by showing that QL-XII-47R, a non-reactive analog, lacks antiviral activity at concentrations more than 20-fold higher than QL-XII-47's IC90. QL-XII-47's inhibition of Zika virus, West Nile virus, hepatitis C virus, and poliovirus further suggests that it acts via a target mediating inhibition of these other medically relevant viruses. These results demonstrate the utility of screens targeting the host reactive cysteinome for rapid identification of compounds with potent antiviral activity.

SIKs control osteocyte responses to parathyroid hormone.

Parathyroid hormone (PTH) activates receptors on osteocytes to orchestrate bone formation and resorption. Here we show that PTH inhibition of SOST (sclerostin), a WNT antagonist, requires HDAC4 and HDAC5, whereas PTH stimulation of RANKL, a stimulator of bone resorption, requires CRTC2. Salt inducible kinases (SIKs) control subcellular localization of HDAC4/5 and CRTC2. PTH regulates both HDAC4/5 and CRTC2 localization via phosphorylation and inhibition of SIK2. Like PTH, new small molecule SIK inhibitors cause decreased phosphorylation and increased nuclear translocation of HDAC4/5 and CRTC2. SIK inhibition mimics many of the effects of PTH in osteocytes as assessed by RNA-seq in cultured osteocytes and following in vivo administration. Once daily treatment with the small molecule SIK inhibitor YKL-05-099 increases bone formation and bone mass. Therefore, a major arm of PTH signalling in osteocytes involves SIK inhibition, and small molecule SIK inhibitors may be applied therapeutically to mimic skeletal effects of PTH.

Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors.

Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.

Development of Chemical Probes for Investigation of Salt-Inducible Kinase Function in Vivo.

Salt-inducible kinases (SIKs) are promising therapeutic targets for modulating cytokine responses during innate immune activation. The study of SIK inhibition in animal models of disease has been limited by the lack of selective small-molecule probes suitable for modulating SIK function in vivo. We used the pan-SIK inhibitor HG-9-91-01 as a starting point to develop improved analogs, yielding a novel probe 5 (YKL-05-099) that displays increased selectivity for SIKs versus other kinases and enhanced pharmacokinetic properties. Well-tolerated doses of YKL-05-099 achieve free serum concentrations above its IC50 for SIK2 inhibition for >16 h and reduce phosphorylation of a known SIK substrate in vivo. While in vivo active doses of YKL-05-099 recapitulate the effects of SIK inhibition on inflammatory cytokine responses, they did not induce metabolic abnormalities observed in Sik2 knockout mice. These results identify YKL-05-099 as a useful probe to investigate SIK function in vivo and further support the development of SIK inhibitors for treatment of inflammatory disorders.

A unique inhibitor binding site in ERK1/2 is associated with slow binding kinetics.

Activation of the ERK pathway is a hallmark of cancer, and targeting of upstream signaling partners led to the development of approved drugs. Recently, SCH772984 has been shown to be a selective and potent ERK1/2 inhibitor. Here we report the structural mechanism for its remarkable selectivity. In ERK1/2, SCH772984 induces a so-far-unknown binding pocket that accommodates the piperazine-phenyl-pyrimidine decoration. This new binding pocket was created by an inactive conformation of the phosphate-binding loop and an outward tilt of helix αC. In contrast, structure determination of SCH772984 with the off-target haspin and JNK1 revealed two canonical but distinct type I binding modes. Notably, the new binding mode with ERK1/2 was associated with slow binding kinetics in vitro as well as in cell-based assay systems. The described binding mode of SCH772984 with ERK1/2 enables the design of a new type of specific kinase inhibitors with prolonged on-target activity.

Discovery of a potent, covalent BTK inhibitor for B-cell lymphoma.

BTK is a member of the TEC family of non-receptor tyrosine kinases whose deregulation has been implicated in a variety of B-cell-related diseases. We have used structure-based drug design in conjunction with kinome profiling and cellular assays to develop a potent, selective, and irreversible BTK kinase inhibitor, QL47, which covalently modifies Cys481. QL47 inhibits BTK kinase activity with an IC50 of 7 nM, inhibits autophosphorylation of BTK on Tyr223 in cells with an EC50 of 475 nM, and inhibits phosphorylation of a downstream effector PLCγ2 (Tyr759) with an EC50 of 318 nM. In Ramos cells QL47 induces a G1 cell cycle arrest that is associated with pronounced degradation of BTK protein. QL47 inhibits the proliferation of B-cell lymphoma cancer cell lines at submicromolar concentrations.