Pyrrolopyrimidine based CSF1R inhibitors: Attempted departure from Flatland.

The colony-stimulating factor 1 receptor (CSF1R) is an attractive target for inflammation disorders and cancers. Based on a series of pyrrolo[2,3-d]pyrimidine containing two carbo-aromatic rings, we have searched for new CSF1R inhibitors having a higher fraction of sp3-atoms. The phenyl unit in the 4-amino group could efficiently be replaced by tetrahydropyran (THP) retaining inhibitor potency. Exchanging the 6-aryl group with cyclohex-2-ene units also resulted in highly potent compounds, while fully saturated ring systems at C-6 led to a loss of activity. The structure-activity relationship study evaluating THP containing pyrrolo[2,3-d]pyrimidine derivates identified several highly active inhibitors by enzymatic studies. A comparison of 11 pairs of THP and aromatic compounds showed that inhibitors containing THP had clear benefits in terms of enzymatic potency, solubility, and cell toxicity. Guided by cellular experiments in Ba/F3 cells, five CSF1R inhibitors were further profiled in ADME assays, indicating the para-aniline derivative 16t as the most attractive compound for further development.

Synthesis and Development of Highly Selective Pyrrolo[2,3-d]pyrimidine CSF1R Inhibitors Targeting the Autoinhibited Form.

Colony-stimulating factor-1 receptor (CSF1R) is a receptor tyrosine kinase that controls the differentiation and maintenance of most tissue-resident macrophages, and the inhibition of CSF1R has been suggested as a possible therapy for a range of human disorders. Herein, we present the synthesis, development, and structure-activity relationship of a series of highly selective pyrrolo[2,3-d]pyrimidines, showing subnanomolar enzymatic inhibition of this receptor and with excellent selectivity toward other kinases in the platelet-derived growth factor receptor (PDGFR) family. The crystal structure of the protein and 23 revealed that the binding conformation of the protein is DFG-out-like. The most promising compounds in this series were profiled for cellular potency and subjected to pharmacokinetic profiling and in vivo stability, indicating that this compound class could be relevant in a potential disease setting. Additionally, these compounds inhibited primarily the autoinhibited form of the receptor, contrasting the behavior of pexidartinib, which could explain the exquisite selectivity of these structures.

White Paper: Mimetics of Class 2 Tumor Suppressor Proteins as Novel Drug Candidates for Personalized Cancer Therapy.

The aim of our proposed concept is to find new target structures for combating cancers with unmet medical needs. This, unfortunately, still applies to the majority of the clinically most relevant tumor entities such as, for example, liver cancer, pancreatic cancer, and many others. Current target structures almost all belong to the class of oncogenic proteins caused by tumor-specific genetic alterations, such as activating mutations, gene fusions, or gene amplifications, often referred to as cancer "driver alterations" or just "drivers." However, restoring the lost function of tumor suppressor genes (TSGs) could also be a valid approach to treating cancer. TSG-derived proteins are usually considered as control systems of cells against oncogenic properties; thus, they represent the brakes in the "car-of-life." Restoring these tumor-defective brakes by gene therapy has not been successful so far, with a few exceptions. It can be assumed that most TSGs are not being inactivated by genetic alteration (class 1 TSGs) but rather by epigenetic silencing (class 2 TSGs or short "C2TSGs"). Reactivation of C2TSGs in cancer therapy is being addressed by the use of DNA demethylating agents and histone deacetylase inhibitors which act on the whole cancer cell genome. These epigenetic therapies have neither been particularly successful, probably because they are "shotgun" approaches that, although acting on C2TSGs, may also reactivate epigenetically silenced oncogenic sequences in the genome. Thus, new strategies are needed to exploit the therapeutic potential of C2TSGs, which have also been named DNA methylation cancer driver genes or "DNAme drivers" recently. Here we present a concept for a new translational and therapeutic approach that focuses on the phenotypic imitation ("mimesis") of proteins encoded by highly disease-relevant C2TSGs/DNAme drivers. Molecular knowledge on C2TSGs is used in two complementary approaches having the translational concept of defining mimetic drugs in common: First, a concept is presented how truncated and/or genetically engineered C2TSG proteins, consisting solely of domains with defined tumor suppressive function can be developed as biologicals. Second, a method is described for identifying small molecules that can mimic the effect of the C2TSG protein lost in the cancer cell. Both approaches should open up a new, previously untapped discovery space for anticancer drugs.

Small-molecule inhibitors of human mitochondrial DNA transcription.

Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system1-6. The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS7-17, and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.

Anti-leukemic effect of CDK9 inhibition in T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is an aggressive malignancy characterized by chemotherapy resistance and a median survival of less than 2 years. Here, we investigated the pharmacological effects of the novel highly specific cyclin-dependent kinase 9 (CDK9) inhibitor LDC526 and its clinically used derivate atuveciclib employing primary T-PLL cells in an ex vivo drug sensitivity testing platform. Importantly, all T-PLL samples were sensitive to CDK9 inhibition at submicromolar concentrations, while conventional cytotoxic drugs were found to be largely ineffective. At the cellular level LDC526 inhibited the phosphorylation at serine 2 of the RNA polymerase II C-terminal domain resulting in decreased de novo RNA transcription. LDC526 induced apoptotic leukemic cell death through down-regulating MYC and MCL1 both at the mRNA and protein level. Microarray-based transcriptomic profiling revealed that genes down-modulated in response to CDK9 inhibition were enriched for MYC and JAK-STAT targets. By contrast, CDK9 inhibition increased the expression of the tumor suppressor FBXW7, which may contribute to decreased MYC and MCL1 protein levels. Finally, the combination of atuvecliclib and the BCL2 inhibitor venetoclax exhibited synergistic anti-leukemic activity, providing the rationale for a novel targeted-agent-based treatment of T-PLL.

Gasdermin D plays a vital role in the generation of neutrophil extracellular traps.

The death of a cell is an inevitable part of its biology. During homeostasis, most cells die through apoptosis. If homeostasis is disturbed, cell death can switch to proinflammatory forms of death, such as necroptosis, pyroptosis, or NETosis. We demonstrate that the formation of neutrophil extracellular traps (NETs), a special form of neutrophil cell death that releases chromatin structures to the extracellular space, is dependent on gasdermin D (GSDMD). GSDMD is a pore-forming protein and an executor of pyroptosis. We screened a chemical library and found a small molecule based on the pyrazolo-oxazepine scaffold that efficiently blocks NET formation and GSDMD-mediated pyroptotic cell death in human cells. During NETosis, GSDMD is proteolytically activated by neutrophil proteases and, in turn, affects protease activation and nuclear expansion in a feed-forward loop. In addition to the central role of GSDMD in pyroptosis, we propose that GSDMD also plays an essential function in NETosis.

Dynarrestin, a Novel Inhibitor of Cytoplasmic Dynein.

Aberrant hedgehog (Hh) signaling contributes to the pathogenesis of multiple cancers. Available inhibitors target Smoothened (Smo), which can acquire mutations causing drug resistance. Thus, compounds that inhibit Hh signaling downstream of Smo are urgently needed. We identified dynarrestin, a novel inhibitor of cytoplasmic dyneins 1 and 2. Dynarrestin acts reversibly to inhibit cytoplasmic dynein 1-dependent microtubule binding and motility in vitro without affecting ATP hydrolysis. It rapidly and reversibly inhibits endosome movement in living cells and perturbs mitosis by inducing spindle misorientation and pseudoprometaphase delay. Dynarrestin reversibly inhibits cytoplasmic dynein 2-dependent intraflagellar transport (IFT) of the cargo IFT88 and flux of Smo within cilia without interfering with ciliogenesis and suppresses Hh-dependent proliferation of neuronal precursors and tumor cells. As such, dynarrestin is a valuable tool for probing cytoplasmic dynein-dependent cellular processes and a promising compound for medicinal chemistry programs aimed at development of anti-cancer drugs.

SAMHD1 is recurrently mutated in T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is an aggressive malignancy with a median survival of the patients of less than two years. Besides characteristic chromosomal translocations, frequent mutations affect the ATM gene, JAK/STAT pathway members, and epigenetic regulators. We here performed a targeted mutation analysis for 40 genes selected from a RNA sequencing of 10 T-PLL in a collection of 28 T-PLL, and an exome analysis of five further cases. Nonsynonymous mutations were identified in 30 of the 40 genes, 18 being recurrently mutated. We identified recurrently mutated genes previously unknown to be mutated in T-PLL, which are SAMHD1, HERC1, HERC2, PRDM2, PARP10, PTPRC, and FOXP1. SAMHD1 regulates cellular deoxynucleotide levels and acts as a potential tumor suppressor in other leukemias. We observed destructive mutations in 18% of cases as well as deletions in two further cases. Taken together, we identified additional genes involved in JAK/STAT signaling (PTPRC), epigenetic regulation (PRDM2), or DNA damage repair (SAMHD1, PARP10, HERC1, and HERC2) as being recurrently mutated in T-PLL. Thus, our study considerably extends the picture of pathways involved in molecular pathogenesis of T-PLL and identifies the tumor suppressor gene SAMHD1 with ~20% of T-PLL affected by destructive lesions likely as major player in T-PLL pathogenesis.

Identification of an (-)-englerin A analogue, which antagonizes (-)-englerin A at TRPC1/4/5 channels.

BACKGROUND AND PURPOSE: (-)-Englerin A (EA) is a potent cytotoxic agent against renal carcinoma cells. It achieves its effects by activation of transient receptor potential canonical (TRPC)4/TRPC1 heteromeric channels. It is also an agonist at channels formed by the related protein, TRPC5. Here, we sought an EA analogue, which might enable a better understanding of these effects of EA. EXPERIMENTAL APPROACH: An EA analogue, A54, was synthesized by chemical elaboration of EA. The effects of EA and A54 on the activity of human TRPC4 or TRPC5 channels overexpressed on A498 and HEK 293 cells were investigated, firstly, by measuring intracellular Ca2+ and, secondly, current using whole-cell patch clamp recordings. KEY RESULTS: A54 had weak or no agonist activity at endogenous TRPC4/TRPC1 channels in A498 cells or TRPC4 or TRPC5 homomeric channels overexpressed in HEK 293 cells. A54 strongly inhibited EA-mediated activation of TRPC4/TRPC1 or TRPC5 and weakly inhibited activation of TRPC4. Studies of TRPC5 showed that A54 shifted the EA concentration-response curve to the right without changing its slope, consistent with competitive antagonism. In contrast, Gd3+ -activated TRPC5 or sphingosine-1-phosphate-activated TRPC4 channels were not inhibited but potentiated by A54. A54 did not activate TRPC3 channels or affect the activation of these channels by the agonist 1-oleoyl-2-acetyl-sn-glycerol. CONCLUSIONS AND IMPLICATIONS: This study has revealed a new tool compound for EA and TRPC1/4/5 channel research, which could be useful for characterizing endogenous TRPC1/4/5 channels and understanding EA-binding sites and their physiological relevance.

Systematic Kinase Inhibitor Profiling Identifies CDK9 as a Synthetic Lethal Target in NUT Midline Carcinoma.

Kinase inhibitors represent the backbone of targeted cancer therapy, yet only a limited number of oncogenic drivers are directly druggable. By interrogating the activity of 1,505 kinase inhibitors, we found that BRD4-NUT-rearranged NUT midline carcinoma (NMC) cells are specifically killed by CDK9 inhibition (CDK9i) and depend on CDK9 and Cyclin-T1 expression. We show that CDK9i leads to robust induction of apoptosis and of markers of DNA damage response in NMC cells. While both CDK9i and bromodomain inhibition over time result in reduced Myc protein expression, only bromodomain inhibition induces cell differentiation and a p21-induced cell-cycle arrest in these cells. Finally, RNA-seq and ChIP-based analyses reveal a BRD4-NUT-specific CDK9i-induced perturbation of transcriptional elongation. Thus, our data provide a mechanistic basis for the genotype-dependent vulnerability of NMC cells to CDK9i that may be of relevance for the development of targeted therapies for NMC patients.

Discovery of a Novel Inhibitor of the Hedgehog Signaling Pathway through Cell-based Compound Discovery and Target Prediction.

Cell-based assays enable monitoring of small-molecule bioactivity in a target-agnostic manner and help uncover new biological mechanisms. Subsequent identification and validation of the small-molecule targets, typically employing proteomics techniques, is very challenging and limited, in particular if the targets are membrane proteins. Herein, we demonstrate that the combination of cell-based bioactive-compound discovery with cheminformatic target prediction may provide an efficient approach to accelerate the process and render target identification and validation more efficient. Using a cell-based assay, we identified the pyrazolo-imidazole smoothib as a new inhibitor of hedgehog (Hh) signaling and an antagonist of the protein smoothened (SMO) with a novel chemotype. Smoothib targets the heptahelical bundle of SMO, prevents its ciliary localization, reduces the expression of Hh target genes, and suppresses the growth of Ptch+/- medulloblastoma cells.

Chemical proteomic analysis reveals alternative modes of action for pyrido[2,3-d]pyrimidine kinase inhibitors.

Small molecule inhibitors belonging to the pyrido[2,3-d]pyrimidine class of compounds were developed as antagonists of protein tyrosine kinases implicated in cancer progression. Derivatives from this compound class are effective against most of the imatinib mesylate-resistant BCR-ABL mutants isolated from advanced chronic myeloid leukemia patients. Here, we established an efficient proteomics method employing an immobilized pyrido[2,3-d]pyrimidine ligand as an affinity probe and identified more than 30 human protein kinases affected by this class of compounds. Remarkably, in vitro kinase assays revealed that the serine/threonine kinases Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK) and p38alpha were among the most potently inhibited kinase targets. Thus, pyrido[2,3-d]pyrimidines did not discriminate between tyrosine and serine/threonine kinases. Instead, we found that these inhibitors are quite selective for protein kinases possessing a conserved small amino acid residue such as threonine at a critical site of the ATP binding pocket. We further demonstrated inhibition of both p38 and RICK kinase activities in intact cells upon pyrido[2,3-d]pyrimidine inhibitor treatment. Moreover, the established functions of these two kinases as signal transducers of inflammatory responses could be correlated with a potent in vivo inhibition of cytokine production by a pyrido[2,3-d]pyrimidine compound. Thus, our data demonstrate the utility of proteomic methods employing immobilized kinase inhibitors for identifying new targets linked to previously unrecognized therapeutic applications.

RICK activates a NF-kappaB-dependent anti-human cytomegalovirus response.

The adapter kinase receptor interacting protein-like interacting caspase-like apoptosis regulatory protein kinase (RICK, also called RIP2 and CARDIAK) was found to be elevated at both the protein and RNA levels during human cytomegalovirus (HCMV) replication, suggesting either that the virus may require RICK for replication or that RICK is part of an unsuccessful host attempt to inhibit HCMV replication. It is demonstrated here that forced expression of RICK in either a kinase active or inactive form activates nuclear factor (NF)-kappaB by means of its intermediate domain and potently blocks HCMV replication in human fibroblasts. Importantly, NF-kappaB activation, which exerted a modestly positive effect on the early phase of infection, clearly had a strongly negative impact during later viral steps. A stable inhibitor of NF-kappaB (IkappaB) reverses the RICK inhibitory effect, and activation of NF-kappaB by IkappaB kinase beta expression is inhibitory to HCMV, demonstrating that NF-kappaB activation is part of a potent anti-HCMV response. Supernatant transfer experiments identified interferon-beta as a downstream component of the RICK inhibitory pathway. RICK expression was found to synergize with HCMV infection in the induction of interferon-beta expression. This study identifies an endogenous RICK-activated, NF-kappaB- and interferon-beta-dependent antiviral pathway that is either inhibited or faulty under normal HCMV replication conditions; efforts to bolster this pathway may lead to novel anti-viral approaches.

An efficient proteomics method to identify the cellular targets of protein kinase inhibitors.

Small molecule inhibitors of protein kinases are widely used in signal transduction research and are emerging as a major class of drugs. Although interpretation of biological results obtained with these reagents critically depends on their selectivity, efficient methods for proteome-wide assessment of kinase inhibitor selectivity have not yet been reported. Here, we address this important issue and describe a method for identifying targets of the widely used p38 kinase inhibitor SB 203580. Immobilization of a suitable SB 203580 analogue and thoroughly optimized biochemical conditions for affinity chromatography permitted the dramatic enrichment and identification of several previously unknown protein kinase targets of SB 203580. In vitro kinase assays showed that cyclin G-associated kinase (GAK) and CK1 were almost as potently inhibited as p38alpha whereas RICK [Rip-like interacting caspase-like apoptosis-regulatory protein (CLARP) kinase/Rip2/CARDIAK] was even more sensitive to inhibition by SB 203580. The cellular kinase activity of RICK, a known signal transducer of inflammatory responses, was already inhibited by submicromolar concentrations of SB 203580 in intact cells. Therefore, our results warrant a reevaluation of the vast amount of data obtained with SB 203580 and might have significant implications on the development of p38 inhibitors as antiinflammatory drugs. Based on the procedures described here, efficient affinity purification techniques can be developed for other protein kinase inhibitors, providing crucial information about their cellular modes of action.

Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein.

Phosphorylation of hepatitis B virus (HBV) core protein has recently been shown to be a prerequisite for pregenomic RNA encapsidation into viral capsids, but the host cell kinases mediating this essential step of the HBV replication cycle have not been identified. We detected two kinases of 95 and 115 kDa in HuH-7 total cell lysates which interacted specifically with the HBV core protein and phosphorylated its arginine-rich C-terminal domain. The 95-kDa kinase was purified and characterized as SR protein-specific kinase 1 (SRPK1) by mass spectrometry. Based on this finding, the 115-kDa kinase could be identified as the related kinase SRPK2 by immunoblot analysis. In vitro, both SRPKs phosphorylated HBV core protein on the same serine residues which are found to be phosphorylated in vivo. Moreover, the major cellular HBV core kinase activity detected in the total cell lysate showed biochemical properties identical to those of SRPK1 and SRPK2, as examined by measuring binding to a panel of chromatography media. We also clearly demonstrate that neither the cyclin-dependent kinases Cdc2 and Cdk2 nor protein kinase C, previously implicated in HBV core protein phosphorylation, can account for the HBV core protein kinase activity. We conclude that both SRPK1 and SRPK2 are most likely the cellular protein kinases mediating HBV core protein phosphorylation during viral infection and therefore represent important host cell targets for therapeutic intervention in HBV infection.