The discovery of a novel pyrrolo[2,3-b]pyridine as a selective CDK8 inhibitor offers a new approach against psoriasis.

Currently, the drugs used in clinical to treat psoriasis mainly broadly suppress cellular immunity. However, these drugs can only provide temporary and partial symptom relief, they do not cure the condition and may lead to recurrence or even serious toxic side effects. In this study, we describe the discovery of a novel potent CDK8 inhibitor as a treatment for psoriasis. Through structure-based design, compound 46 was identified as the most promising candidate, exhibiting a strong inhibitory effect on CDK8 (IC50 value of 57 nM) along with favourable inhibition against NF-κB. Additionally, it demonstrated a positive effect in an in vitro psoriasis model induced by TNF-α. Furthermore, this compound enhanced the thermal stability of CDK8 and exerted evident effects on the biological function of CDK8, and it had favourable selectivity across the CDK family and tyrosine kinase. This compound showed no obvious inhibitory effect on CYP450 enzyme. Further studies confirmed that compound 46 exhibited therapeutic effect on IMQ-induced psoriasis, alleviated the inflammatory response in mice, and enhanced the expression of Foxp3 and IL-10 in the dorsal skin in vivo. This discovery provides a new strategy for developing selective CDK8 inhibitors with anti-inflammatory activity for the treatment of psoriasis.

Discovery of a Novel and Potent Cyclin-Dependent Kinase 8/19 (CDK8/19) Inhibitor for the Treatment of Cancer.

The mediator kinases CDK8 and CDK19 control the dynamic transcription of selected genes in response to various signals and have been shown to be hijacked to sustain hyperproliferation by various solid and liquid tumors. CDK8/19 is emerging as a promising anticancer therapeutic target. Here, we report the discovery of compound 12, a novel small molecule CDK8/19 inhibitor. This molecule demonstrated not only decent enzymatic and cellular activities but also remarkable selectivity in CDK and kinome panels. Besides, compound 12 also displayed favorable ADME profiles including low CYP1A2 inhibition, acceptable clearance, and high oral bioavailability in multiple preclinical species. Robust in vivo PD and efficacy studies in mice models further demonstrated its potential use as mono- and combination therapy for the treatment of cancers.

An ensemble machine learning model generates a focused screening library for the identification of CDK8 inhibitors.

The identification of an effective inhibitor is an important starting step in drug development. Unfortunately, many issues such as the characterization of protein binding sites, the screening library, materials for assays, etc., make drug screening a difficult proposition. As the size of screening libraries increases, more resources will be inefficiently consumed. Thus, new strategies are needed to preprocess and focus a screening library towards a targeted protein. Herein, we report an ensemble machine learning (ML) model to generate a CDK8-focused screening library. The ensemble model consists of six different algorithms optimized for CDK8 inhibitor classification. The models were trained using a CDK8-specific fragment library along with molecules containing CDK8 activity. The optimized ensemble model processed a commercial library containing 1.6 million molecules. This resulted in a CDK8-focused screening library containing 1,672 molecules, a reduction of more than 99.90%. The CDK8-focused library was then subjected to molecular docking, and 25 candidate compounds were selected. Enzymatic assays confirmed six CDK8 inhibitors, with one compound producing an IC50 value of ≤100 nM. Analysis of the ensemble ML model reveals the role of the CDK8 fragment library during training. Structural analysis of molecules reveals the hit compounds to be structurally novel CDK8 inhibitors. Together, the results highlight a pipeline for curating a focused library for a specific protein target, such as CDK8.

Mediator kinase inhibition reverses castration resistance of advanced prostate cancer.

Mediator kinases CDK19 and CDK8, pleiotropic regulators of transcriptional reprogramming, are differentially regulated by androgen signaling, but both kinases are upregulated in castration-resistant prostate cancer (CRPC). Genetic or pharmacological inhibition of CDK8 and CDK19 reverses the castration-resistant phenotype and restores the sensitivity of CRPC xenografts to androgen deprivation in vivo. Prolonged CDK8/19 inhibitor treatment combined with castration not only suppressed the growth of CRPC xenografts but also induced tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplified and modulated the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affected stromal gene expression, indicating that Mediator kinase activity in CRPC molded the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulated the MYC pathway, and Mediator kinase inhibition suppressed a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors showed efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlated with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediated androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.

The study of a novel CDK8 inhibitor E966-0530-45418 that inhibits prostate cancer metastasis in vitro and in vivo.

Prostate cancer is a prevalent malignancy among men globally, and androgen deprivation therapy is the conventional first-line treatment for metastatic prostate cancer. While androgen deprivation therapy is efficacious in castration-sensitive prostate cancer, it remains less effective in castration-resistant cases. Transcriptional dysregulation is a well-established hallmark of cancer, and targeting proteins involved in transcriptional regulation, such as cyclin-dependent kinase 8 (CDK8), has become an attractive therapeutic strategy. CDK8, a nuclear serine-threonine kinase, is a key component of the mediator complex and plays a critical role in transcriptional regulation. Recent studies have highlighted the promising role of CDK8 as a target in the treatment of metastatic prostate cancer. Our study assessed the efficacy of a novel CDK8 inhibitor, E966-0530-45418, which exhibited potent CDK8 inhibition (IC50 of 129 nM) and high CDK8 selectivity. Treatment with E966-0530-45418 significantly inhibited prostate cancer cell migration and epithelial-to-mesenchymal transition (EMT) at both the RNA and protein levels. Further mechanistic analysis indicated that E966-0530-45418 suppresses prostate cancer metastasis by decreasing CDK8 activity and inhibiting TGF-ß1-mediated Smad3/RNA polymerase II linker phosphorylation and Akt/GSK3ß/ß-catenin signaling. The results in animal model also showed that E966-0530-45418 exhibited anti-metastatic properties in vivo. Our study demonstrated that E966-0530-45418 has great therapeutic potential in the treatment of metastatic prostate cancer.

A novel CDK8 inhibitor with poly-substituted pyridine core: Discovery and anti-inflammatory activity evaluation in vivo.

As an ideal anti-inflammatory target, cyclin-dependent kinase 8 (CDK8) has gradually attracted the attention of researchers. CDK8 inhibition up-regulates Interleukin-10 (IL-10) expression by enhancing the transcriptional activity of activator protein-1 (AP-1), and augmenting IL-10 abundance is a viable strategy for the treatment of inflammatory bowel disease (IBD). In this research, through structure-based drug design and dominant fragment hybridization, a series of poly-substituted pyridine derivatives were designed and synthesized as CDK8 inhibitors. Ultimately, compound CR16 was identified as the best one, which exhibited good inhibitory activity against CDK8 (IC50 = 74.4 nM). In vitro and in vivo studies indicated that CR16 could enhance the transcriptional activity of AP-1, augment the abundance of IL-10, and affect CDK8-related signaling pathways including TLR7/NF-κB/MAPK and IL-10-JAK1-STAT3 pathways. In addition, CR16 showed potent therapeutic effect in an animal model of IBD.

MK256 is a novel CDK8 inhibitor with potent antitumor activity in AML through downregulation of the STAT pathway.

Acute myeloid leukemia (AML) is the most lethal form of AML due to disease relapse. Cyclin dependent kinase 8 (CDK8) is a serine/threonine kinase that belongs to the family of Cyclin-dependent kinases and is an emerging target for the treatment of AML. MK256, a potent, selective, and orally available CDK8 inhibitor was developed to target AML. We sought to examine the anticancer effect of MK256 on AML. In CD34+/CD38- leukemia stem cells, we found that MK256 induced differentiation and maturation. Treatment of MK256 inhibited proliferation of AML cell lines. Further studies of the inhibitory effect suggested that MK256 not only downregulated phosphorylated STAT1(S727) and STAT5(S726), but also lowered mRNA expressions of MCL-1 and CCL2 in AML cell lines. Efficacy of MK256 was shown in MOLM-14 xenograft models, and the inhibitory effect on phosphorylated STAT1(S727) and STAT5(S726) with treatment of MK256 was observed in vivo. Pharmacologic dynamics study of MK256 in MOLM-14 xenograft models showed dose-dependent inhibition of the STAT pathway. Both in vitro and in vivo studies suggested that MK256 could effectively downregulate the STAT pathway. In vitro ADME, pharmacological kinetics, and toxicity of MK256 were profiled to evaluate the drug properties of MK256. Our results show that MK256 is a novel CDK8 inhibitor with a desirable efficacy and safety profile and has great potential to be a promising drug candidate for AML through regulating the STAT pathway.

A Selective and Orally Bioavailable Quinoline-6-Carbonitrile-Based Inhibitor of CDK8/19 Mediator Kinase with Tumor-Enriched Pharmacokinetics.

Senexins are potent and selective quinazoline inhibitors of CDK8/19 Mediator kinases. To improve their potency and metabolic stability, quinoline-based derivatives were designed through a structure-guided strategy based on the simulated drug-target docking model of Senexin A and Senexin B. A library of quinoline-Senexin derivatives was synthesized to explore the structure-activity relationship (SAR). An optimized compound 20a (Senexin C) exhibits potent CDK8/19 inhibitory activity with high selectivity. Senexin C is more metabolically stable and provides a more sustained inhibition of CDK8/19-dependent cellular gene expression when compared with the prototype inhibitor Senexin B. In vivo pharmacokinetic (PK) and pharmacodynamic (PD) evaluation using a novel tumor-based PD assay showed good oral bioavailability of Senexin C with a strong tumor-enrichment PK profile and tumor-PD marker responses. Senexin C inhibits MV4-11 leukemia growth in a systemic in vivo model with good tolerability.

Discovery of a novel cyclin-dependent kinase 8 inhibitor with an oxindole core for anti-inflammatory treatment.

Chronic inflammation is an underlying cause in a number of diseases. Cyclin-dependent kinase 8 (CDK8) has been implicated as an inflammatory mediator, indicating its potential as an anti-inflammatory target. Herein, we performed structure-based virtual screening (SBVS) to identify novel CDK8 inhibitors. The pharmacological interactions for CDK8 were identified and incorporated into a SBVS protocol. Selected compounds were tested in enzymatic assays, and one compound was confirmed to be a CDK8 inhibitor with a 50% inhibitory concentration (IC50) value of 1684.4 nM. Comparing structural analogs identified a compound, F059-1017, with greater potency (IC50 558.1 nM). When tested in cell lines, the compounds displayed low cytotoxicity. Cellular assays revealed that the identified CDK8 inhibitors can reduce phosphorylation and expression of signaling mediators associated with inflammation. In addition, results of kinase profiling showed that compound F059-1017 is selective towards CDK8. These findings suggest that the new inhibitors have great potential as lead compounds for developing novel anti-inflammatory therapeutics.

The Cyclin-Dependent Kinase 8 (CDK8) Inhibitor DCA Promotes a Tolerogenic Chemical Immunophenotype in CD4+ T Cells via a Novel CDK8-GATA3-FOXP3 Pathway.

Immune health requires innate and adaptive immune cells to engage precisely balanced pro- and anti-inflammatory forces. We employ the concept of chemical immunophenotypes to classify small molecules functionally or mechanistically according to their patterns of effects on primary innate and adaptive immune cells. The high-specificity, low-toxicity cyclin-dependent kinase 8 (CDK8) inhibitor 16-didehydro-cortistatin A (DCA) exerts a distinct tolerogenic profile in both innate and adaptive immune cells. DCA promotes regulatory T cells (Treg) and Th2 differentiation while inhibiting Th1 and Th17 differentiation in both murine and human cells. This unique chemical immunophenotype led to mechanistic studies showing that DCA promotes Treg differentiation in part by regulating a previously undescribed CDK8-GATA3-FOXP3 pathway that regulates early pathways of Foxp3 expression. These results highlight previously unappreciated links between Treg and Th2 differentiation and extend our understanding of the transcription factors that regulate Treg differentiation and their temporal sequencing. These findings have significant implications for future mechanistic and translational studies of CDK8 and CDK8 inhibitors.

Identification of 3, 4-disubstituted pyridine derivatives as novel CDK8 inhibitors.

Selective inhibition of cyclin-dependent kinase 8 (CDK8) has been recently regarded as a potential approach for cancer therapy. A series of novel CDK8 inhibitors with the pyridine core was identified via scaffold hopping from the known CDK8 inhibitor A-7. The new inhibitors were designed to improve the ligand efficiency so as to enhance drug-likeness. Most of the compounds showed significant inhibition against CDK8/cyclin C, and the most active compounds (5d, 5e and 7') displayed IC50 values of 2.4 nM, 5.0 nM and 7.7 nM, respectively. Preliminary kinase profiling of selected compounds against a panel of kinases from different families indicated that this compound class might selectively inhibit CDK8 as well as its paralog CDK19. Some compounds exhibited cellular activity in both MTT and SRB assays against a variety of tumor cells, including HCT-116, A549, MDA-MB-231, KB, KB-VIN and MCF-7. Further flow cytometry analysis revealed a dose-dependent G2/M phase arrest in MDA-MB-231 cells treated with compounds 6'a, 6'b, 6'j and 6'k. In addition, compound 6'k demonstrated moderate antitumor efficacy in HCT-116 mouse models, although unfavorable pharmacokinetic profiles were suggested by preliminary study in mice. The results provided a new structural prototype for the search of selective CDK8 inhibitors as antitumor agents.

Stability of Imprinting and Differentiation Capacity in Naïve Human Cells Induced by Chemical Inhibition of CDK8 and CDK19.

Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naïve and primed states are the best characterized pluripotency states. Naïve pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naïve PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naïve human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naïve human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naïve human PSCs. We report that long-term cultured CDK8/19i-naïve human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naïve human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naïve state. Interestingly, long-term cultured CDK8/19i-naïve human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naïve human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naïve state that preserves imprinting and potency over long-term culture.

Discovery of a potent, highly selective, and orally bioavailable inhibitor of CDK8 through a structure-based optimisation.

CDK8 is deregulated in multiple types of human cancer and is viewed as a therapeutic target for the treatment of the disease. Accordingly, the search for small-molecule inhibitors of CDK8 is being intensified. Capitalising on our initial discovery of AU1-100, a potent CDK8 inhibitor yet with a limited degree of kinase selectivity, a structure-based optimisation was carried out, with a series of new multi-substituted pyridines rationally designed, chemically prepared and biologically evaluated. Such endeavour has culminated in the identification of 42, a more potent CDK8 inhibitor with superior kinomic selectivity and oral bioavailability. The mechanism underlying the anti-proliferative effect of 42 on MV4-11 cells was studied, revealing that the compound arrested the G1 cell cycle and triggered apoptosis. The low risk of hepato- and cardio-toxicity of 42 was estimated. These findings merit further investigation of 42 as a targeted cancer therapeutic.

Dissection of two routes to naïve pluripotency using different kinase inhibitors.

Embryonic stem cells (ESCs) can be maintained in the naïve state through inhibition of Mek1/2 and Gsk3 (2i). A relevant effect of 2i is the inhibition of Cdk8/19, which are negative regulators of the Mediator complex, responsible for the activity of enhancers. Inhibition of Cdk8/19 (Cdk8/19i) stimulates enhancers and, similar to 2i, stabilizes ESCs in the naïve state. Here, we use mass spectrometry to describe the molecular events (phosphoproteome, proteome, and metabolome) triggered by 2i and Cdk8/19i on ESCs. Our data reveal widespread commonalities between these two treatments, suggesting overlapping processes. We find that post-transcriptional de-repression by both 2i and Cdk8/19i might support the mitochondrial capacity of naive cells. However, proteome reprogramming in each treatment is achieved by different mechanisms. Cdk8/19i acts directly on the transcriptional machinery, activating key identity genes to promote the naïve program. In contrast, 2i stabilizes the naïve circuitry through, in part, de-phosphorylation of downstream transcriptional effectors.

Potent and orally bioavailable CDK8 inhibitors: Design, synthesis, structure-activity relationship analysis and biological evaluation.

CDK8 regulates transcription either by phosphorylation of transcription factors or, as part of a four-subunit kinase module, through a reversible association of the kinase module with the Mediator complex, a highly conserved transcriptional coactivator. Deregulation of CDK8 has been found in various types of human cancer, while the role of CDK8 in supressing anti-cancer response of natural killer cells is being understood. Currently, CDK8-targeting cancer drugs are highly sought-after. Herein we detail the discovery of a series of novel pyridine-derived CDK8 inhibitors. Medicinal chemistry optimisation gave rise to 38 (AU1-100), a potent CDK8 inhibitor with oral bioavailability. The compound inhibited the proliferation of MV4-11 acute myeloid leukaemia cells with the kinase activity of cellular CDK8 dampened. No systemic toxicology was observed in the mice treated with 38. These results warrant further pre-clinical studies of 38 as an anti-cancer agent.

The Inhibition of CDK8/19 Mediator Kinases Prevents the Development of Resistance to EGFR-Targeting Drugs.

Drug resistance is the main obstacle to achieving cures with both conventional and targeted anticancer drugs. The emergence of acquired drug resistance is initially mediated by non-genetic transcriptional changes, which occur at a much higher frequency than mutations and may involve population-scale transcriptomic adaptation. CDK8/19 kinases, through association with transcriptional Mediator complex, regulate transcriptional reprogramming by co-operating with different signal-responsive transcription factors. Here we tested if CDK8/19 inhibition could prevent adaptation to drugs acting on epidermal growth factor receptor (EGFR/ERBB1/HER1). The development of resistance was analyzed following long-term exposure of BT474 and SKBR3 breast cancer cells to EGFR-targeting small molecules (gefitinib, erlotinib) and of SW48 colon cancer cells to an anti-EGFR monoclonal antibody cetuximab. In all cases, treatment of small cell populations (~105 cells) with a single dose of the drug initially led to growth inhibition that was followed by the resumption of proliferation and development of drug resistance in the adapted populations. However, this adaptation was always prevented by the addition of selective CDK8/19 inhibitors, even though such inhibitors alone had only moderate or no effect on cell growth. These results indicate that combining EGFR-targeting drugs with CDK8/19 inhibitors may delay or prevent the development of tumor resistance to therapy.

Loss of Mediator complex subunit 13 (MED13) promotes resistance to alkylation through cyclin D1 upregulation.

Alkylating drugs are among the most often used chemotherapeutics. While cancer cells frequently develop resistance to alkylation treatments, detailed understanding of mechanisms that lead to the resistance is limited. Here, by using genome-wide CRISPR-Cas9 based screen, we identify transcriptional Mediator complex subunit 13 (MED13) as a novel modulator of alkylation response. The alkylation exposure causes significant MED13 downregulation, while complete loss of MED13 results in reduced apoptosis and resistance to alkylating agents. Transcriptome analysis identified cyclin D1 (CCND1) as one of the highly overexpressed genes in MED13 knock-out (KO) cells, characterized by shorter G1 phase. MED13 is able to bind to CCND1 regulatory elements thus influencing the expression. The resistance of MED13 KO cells is directly dependent on the cyclin D1 overexpression, and its down-regulation is sufficient to re-sensitize the cells to alkylating agents. We further demonstrate the therapeutic potential of MED13-mediated response, by applying combinatory treatment with CDK8/19 inhibitor Senexin A. Importantly, the treatment with Senexin A stabilizes MED13, and in combination with alkylating agents significantly reduces viability of cancer cells. In summary, our findings identify novel alkylation stress response mechanism dependent on MED13 and cyclin D1 that can serve as basis for development of innovative therapeutic strategies.

Angel or Devil ? - CDK8 as the new drug target.

Cyclin-dependent kinase 8 (CDK8) plays an momentous role in transcription regulation by forming kinase module or transcription factor phosphorylation. A large number of evidences have identified CDK8 as an important factor in cancer occurrence and development. In addition, CDK8 also participates in the regulation of cancer cell stress response to radiotherapy and chemotherapy, assists tumor cell invasion, metastasis, and drug resistance. Therefore, CDK8 is regarded as a promising target for cancer therapy. Most studies in recent years supported the role of CDK8 as a carcinogen, however, under certain conditions, CDK8 exists as a tumor suppressor. The functional diversity of CDK8 and its exceptional role in different types of cancer have aroused great interest from scientists but even more controversy during the discovery of CDK8 inhibitors. In addition, CDK8 appears to be an effective target for inflammation diseases and immune system disorders. Therefore, we summarized the research results of CDK8, involving physiological/pathogenic mechanisms and the development status of compounds targeting CDK8, provide a reference for the feasibility evaluation of CDK8 as a therapeutic target, and guidance for researchers who are involved in this field for the first time.

All-Atomic Molecular Dynamic Studies of Human and Drosophila CDK8: Insights into Their Kinase Domains, the LXXLL Motifs, and Drug Binding Site.

Cyclin-dependent kinase 8 (CDK8) and its regulatory partner Cyclin C (CycC) play conserved roles in modulating RNA polymerase II (Pol II)-dependent gene expression. To understand the structure and function relations of CDK8, we analyzed the structures of human and Drosophila CDK8 proteins using molecular dynamics simulations, combined with functional analyses in Drosophila. Specifically, we evaluated the structural differences between hCDK8 and dCDK8 to predict the effects of the LXXLL motif mutation (AQKAA), the P154L mutations, and drug binding on local structures of the CDK8 proteins. First, we have observed that both the LXXLL motif and the kinase activity of CDK8 are required for the normal larval-to-pupal transition in Drosophila. Second, our molecular dynamic analyses have revealed that hCDK8 has higher hydrogen bond occupation of His149-Asp151 and Asp151-Asn156 than dCDK8. Third, the substructure of Asp282, Phe283, Arg285, Thr287 and Cys291 can distinguish human and Drosophila CDK8 structures. In addition, there are two hydrogen bonds in the LXXLL motif: a lower occupation between L312 and L315, and a relatively higher occupation between L312 and L316. Human CDK8 has higher hydrogen bond occupation between L312 and L316 than dCDK8. Moreover, L312, L315 and L316 in the LXXLL motif of CDK8 have the specific pattern of hydrogen bonds and geometries, which could be crucial for the binding to nuclear receptors. Furthermore, the P154L mutation dramatically decreases the hydrogen bond between L312 and L315 in hCDK8, but not in dCDK8. The mutations of P154L and AQKAA modestly alter the local structures around residues 154. Finally, we identified the inhibitor-induced conformational changes of hCDK8, and our results suggest a structural difference in the drug-binding site between hCDK8 and dCDK8. Taken together, these results provide the structural insights into the roles of the LXXLL motif and the kinase activity of CDK8 in vivo.

Pathogenesis of CDK8-associated disorder: two patients with novel CDK8 variants and in vitro and in vivo functional analyses of the variants.

Cyclin-dependent kinase 8 (CDK8) is a member of the CDK/Cyclin module of the mediator complex. A recent study reported that heterozygous missense CDK8 mutations cause a neurodevelopmental disorder in humans. The mechanistic basis of CDK8-related disorder has yet to be delineated. Here, we report 2 patients with de novo missense mutations within the kinase domain of CDK8 along with the results of in vitro and in vivo functional analyses using a zebrafish model. Patient 1 and Patient 2 had intellectual disabilities and congenital anomalies. Exome analyses showed that patient 1 had a heterozygous de novo missense p.G28A variant in the CDK8 (NM_001260.3) gene and patient 2 had a heterozygous de novo missense p.N156S variant in the CDK8 gene. We assessed the pathogenicity of these two variants using cultured-cells and zebrafish model. An in vitro kinase assay of human CDK8 showed that enzymes with a p.G28A or p.N156S substitution showed decreased kinase activity. An in vivo assays of zebrafish overexpression analyses also showed that the p.G28A and p.N156S alleles were hypomorphic alleles. Importantly, the inhibition of CDK8 kinase activity in zebrafish embryos using a specific chemical inhibitor induced craniofacial and heart defects similar to the patients' phenotype. Taken together, zebrafish studies showed that non-synonymous variants in the kinase domain of CDK8 act as hypomorphic alleles causing human congenital disorder.